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  • 1.
    Abidine, Yara
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Liu, Lifeng
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Wallén, Oskar
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Trybala, Edward
    Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
    Olofsson, Sigvard
    Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
    Bergström, Tomas
    Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
    Bally, Marta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Cellular Chondroitin Sulfate and the Mucin-like Domain of Viral Glycoprotein C Promote Diffusion of Herpes Simplex Virus 1 While Heparan Sulfate Restricts Mobility2022In: Viruses, E-ISSN 1999-4915, Vol. 14, no 8, article id 1836Article in journal (Refereed)
    Abstract [en]

    The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

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  • 2.
    Adhikari, Deepak
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Flohr, Gilian
    Hogeschool Leiden, Zernikedreef 11,2333 CK Leiden, The Netherlands.
    Gorre, Nagaraju
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shen, Yan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yang, Hairu
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundin, Eva
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Lan, Zijian
    University of Louisville Health Sciences Center, Louisville, Kentucky, USA.
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Disruption of Tsc2 in oocytes leads to overactivation of the entire pool of primordial follicles2009In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 15, no 12, p. 765-770Article in journal (Refereed)
    Abstract [en]

    To maintain the length of reproductive life in a woman, it is essential that most of her ovarian primordial follicles are maintained in a quiescent state to provide a continuous supply of oocytes. However, our understanding of the molecular mechanisms that control the quiescence and activation of primordial follicles is still in its infancy. In this study, we provide some genetic evidence to show that the tumor suppressor tuberous sclerosis complex 2 (Tsc2), which negatively regulates mammalian target of rapamycin complex 1 (mTORC1), functions in oocytes to maintain the dormancy of primordial follicles. In mutant mice lacking the Tsc2 gene in oocytes, the pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in oocytes. This results in depletion of follicles in early adulthood, causing premature ovarian failure (POF). Our results suggest that the Tsc1-Tsc2 complex mediated suppression of mTORC1 activity is indispensable for maintenance of the dormancy of primordial follicles, thus preserving the follicular pool, and that mTORC1 activity in oocytes promotes follicular activation. Our results also indicate that deregulation of Tsc/mTOR signaling in oocytes may cause pathological conditions of the ovary such as infertility and POF.

  • 3.
    Alam, Md Khorshed
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vinklarek, Ivo
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sachl, Radek
    Fluorescence Studies of Lipid Distribution in Bilayers under Oxidative Stress2019In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, no 3, p. 508A-508AArticle in journal (Other academic)
  • 4. Ans, Muhammad
    et al.
    Iqbal, Javed
    Eliasson, Bertil
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Saif, Muhammad Jawwad
    Javed, Hafiz Muhammad Asif
    Ayub, Khurshid
    Designing of non-fullerene 3D star-shaped acceptors for organic solar cells2019In: Journal of Molecular Modeling, ISSN 1610-2940, E-ISSN 0948-5023, Vol. 25, no 5, article id 129Article in journal (Refereed)
    Abstract [en]

    The design and fabrication of solar cells have recently witnessed the exploration of non-fullerene-based acceptor molecules for higher efficiency. In this study, the optical and electronic properties of four new three-dimensional (3D) star-shaped acceptor molecules (M1, M2, M3, and M4) are evaluated for use as acceptor molecules in organic solar cells. These molecules contain a triphenylamine donor core with diketopyrrolopyrrole acceptor arms linked via a thiophene bridge unit. Molecules M1–M4 are characterized by different end-capped acceptor moieties, including 2-(5-methylene-6-oxo-5,6-dihydrocyclopenta-b-thiophen-4-ylidene)malononitrile (M1), 2-(2-methylene-3-oxo-2,3-dihydroinden-1-ylidene)malononitrile (M2), 2-(5-methyl-2-methylene-3-oxo-2,3-dihydroinden-1-ylidene)malononitrile (M3), and 3-methyl-5-methylnene-thioxothiazolidin-4-one (M4). The properties of the newly designed molecules were compared with a well-known reference compound R, which was recently reported as an excellent acceptor molecule for organic solar cells. Molecules M1–M4 exhibit suitable frontier molecular orbital patterns for charge mobility. M2 shows maximum absorption (λmax) at 846.8 nm in dichloromethane solvent, which is ideal for the design of transparent solar cells. A strong electron withdrawing end-capped acceptor causes a red shift in absorption spectra. All molecules are excellent for hole mobility due to a lower value of λh compared to the reference R.

  • 5. Arbeitman, Claudia R.
    et al.
    Rojas, Pablo
    Ojeda-May, Pedro
    Umeå University, Faculty of Science and Technology, High Performance Computing Center North (HPC2N). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Garcia, Martin E.
    The SARS-CoV-2 spike protein is vulnerable to moderate electric fields2021In: Nature Communications, E-ISSN 2041-1723, Vol. 12, no 1, article id 5407Article in journal (Refereed)
    Abstract [en]

    Most of the ongoing projects aimed at the development of specific therapies and vaccines against COVID-19 use the SARS-CoV-2 spike (S) protein as the main target. The binding of the spike protein with the ACE2 receptor (ACE2) of the host cell constitutes the first and key step for virus entry. During this process, the receptor binding domain (RBD) of the S protein plays an essential role, since it contains the receptor binding motif (RBM), responsible for the docking to the receptor. So far, mostly biochemical methods are being tested in order to prevent binding of the virus to ACE2. Here we show, with the help of atomistic simulations, that external electric fields of easily achievable and moderate strengths can dramatically destabilise the S protein, inducing long-lasting structural damage. One striking field-induced conformational change occurs at the level of the recognition loop L3 of the RBD where two parallel beta sheets, believed to be responsible for a high affinity to ACE2, undergo a change into an unstructured coil, which exhibits almost no binding possibilities to the ACE2 receptor. We also show that these severe structural changes upon electric-field application also occur in the mutant RBDs corresponding to the variants of concern (VOC) B.1.1.7 (UK), B.1.351 (South Africa) and P.1 (Brazil). Remarkably, while the structural flexibility of S allows the virus to improve its probability of entering the cell, it is also the origin of the surprising vulnerability of S upon application of electric fields of strengths at least two orders of magnitude smaller than those required for damaging most proteins. Our findings suggest the existence of a clean physical method to weaken the SARS-CoV-2 virus without further biochemical processing. Moreover, the effect could be used for infection prevention purposes and also to develop technologies for in-vitro structural manipulation of S. Since the method is largely unspecific, it can be suitable for application to other mutations in S, to other proteins of SARS-CoV-2 and in general to membrane proteins of other virus types.

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  • 6.
    Bag, Pushan
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    How could Christmas trees remain evergreen?: photosynthetic acclimation of Scots pine and Norway spruce needles during winter2022Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Plants and other green organisms harvest sunlight by green chlorophyll pigments and covertit to chemical energy (sugars) and oxygen in a process called photosynthesis providing the foundation for life on Earth. Although it is unanimously believed that oceanic phytoplanktons are the main contributors to the global photosynthesis, the contribution of coniferous boreal forests distributed across vast regions of the northern hemisphere cannot be undermined. Hence boreal forests account signifificantly for social, economical and environmental sustainability. Not only do conifers thrive in the tundra regions with extreme climate, but they also maintain their needles green over the boreal winter. A question remains; what makes them so resilient? In this respect, we aimed to understand the remarkable winter adaptation strategies in two dominant boreal coniferous species,i.e., Pinus sylvestris and Picea abies. First, we mapped the transcriptional landscape in Norway spruce (Picea abies) needles over the annual cycle. Transcriptional changes in the nascent needles reflflected a sequence of developmental processes and active vegetative growth during early summer and summer. Later after maturation, transcriptome reflflected activated defense against biotic factors and acclimationin response to abiotic environmental cues such as freezing temperatures during winter. Secondly, by monitoring the photosynthetic performance of Scot pine needles, we found that the trees face extreme stress during the early spring (Feb-Mar) when sub-zero temperatures are accompanied by high solar radiation. At this time, drastic changes occur in the thylakoid membranes of the chloroplast that allows the mixing of photosystem I and photosystem II that typically remain laterally segregated. This triggers direct energy transfer from PSII to PSI and thus protects PSII from damage. Furthermore, we found that this loss of lateral segregation may be a consequence of triple phosphorylationof Lhcb1 (Light harvesting complex1 of photosystem II). The structural changes in thylakoid membranes also lead to changes inthe thylakoid macro domain organisationand pigment protein composition. Furthermore, we discovered that while PSII is protected by direct energy transfer, the protection of PSI is provided through photoreduction of oxygen by flavodiiron proteins, which in turn allows P700 to stay in an oxidised state necessary for direct energy transfer. These coordinated cascades of changes concomitantly protect both PSI and PSII to maintain the needles green over the winter.

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  • 7. Baker, Joseph
    et al.
    Dahlberg, Tobias
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Bullitt, Esther
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress2021In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 118, no 21, article id e2023595118Article in journal (Refereed)
    Abstract [en]

    Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that Class 1a pili of uropathogenic E. coli (UPEC), as well as Class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC Class 5 pili. Examining structural and steered molecular dynamics simulation data, we find this difference in Class 1 pili subunit behavior originates from an alpha-helical motif that can unfold when exposed to force. A disulfide bond cross-linking beta-strands in Class 1 pili stabilizes subunits, allowing them to tolerate higher forces than Class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, Class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of Class 5 ETEC pili.

  • 8. Baker, Joseph
    et al.
    Dahlberg, Tobias
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Bullitt, Esther
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Unveiling the Contributions of Secondary Structure and Disulfide Bonds for Bacterial Adhesion Pili Extension using a Multiscale Approach2021Conference paper (Other academic)
    Abstract [en]

    Bacterial adhesion pili are essential virulence factors for many pathogenic Escherichia coli, including bacteria that cause urinary tract infections (UPEC) and diarrheal diseases (ETEC). To sustain adhesion under forces similar to those in the fluid environments of the urinary tract and gastrointestinal tract, these pili (also called fimbriae) can extend to over seven times their original length. Both UPEC and ETEC can uncoil their quaternary structure under pulling force and re-coil to their helical form when the force is reduced, as observed using optical tweezers. However, after extension to a linear polymer UPEC undergo an additional reversible conformational change, that is not seen in ETEC. The mechanism for this conformational change in UPEC is not known. Therefore, to obtain a comprehensive picture of pilus extension we have taken a synergistic approach that combines optical tweezer experiments, structural data from cryo-EM, and steered molecular dynamics simulations to investigate the response of pilin subunits to force.

    Our multi-faceted approach provides novel molecular-scale insights into the structural changes that occur in UPEC and ETEC pili under pulling forces. We find that the conformational change observed in UPEC pili in optical tweezer experiments is correlated with the presence of an alpha helix. In addition, structural analysis and steered molecular dynamics simulations show that there is a disulfide bond that provides additional stability of UPEC pilin subunits that is not observed in ETEC pilins, which lack cysteine residues. Together, these results suggest that the mechanism of extension of bacterial adhesion pili is related to their environmental niche, and the magnitude of fluid forces in the urinary tract versus the GI tract.

  • 9.
    Bally, Marta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Regulating the dynamic interactions between herpes simplex viruses and cell -surface glycosaminoglycans2019In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 48, p. S41-S41Article in journal (Other academic)
    Abstract [en]

    Virus entry is a complex dynamic multistep process requiring a series of fine-tuned events mediating virus diffusion through the glycocalyx, its attachment to the cell membrane and lateral diffusion to the point of entry. A number of enveloped viruses, including herpes simplex viruses (HSV) attach to susceptible host cells via interaction between their glycoproteins and cell-surface glycosaminoglycans (GAGs). In our work, we study the molecular and physical mechanisms modulating HSV binding, diffusion and release from cell-surface glycosaminoglycans. Using single virus tracking in combination with either in vitro minimal models of the cell surface or live cell microscopy, we gain insights into the modulatory function of protein glycosylation (the presence of mucin-like regions on viral glycoproteins) and interrogate the role of GAG sulfation in the process. We show that mucin-like regions found on the glycoproteins of HSV-1 and HSV-2 play an important role in modulating the interaction, an observation further supported by cell experiments. We further show that the diffusion of virions on the surface depends on the type of GAGs and their degree of sulfation. Taken together, our research contributes to a better understanding of the mechanisms underlying the interaction between a virus and the surface of its host. Such insights will without doubt facilitate the design of more efficient antiviral drugs or vaccines.

  • 10.
    Bally, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Block, Stephan
    Department of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
    Höök, Fredrik
    Department of Physics, Chalmers University of Technology, Gothenburg, Sweden.
    Larson, Göran
    Department of Laboratory Medicine, Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital, Bruna Stråket 16, Gothenburg, Sweden.
    Parveen, Nagma
    Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, India.
    Rydell, Gustaf E.
    Department of Infectious Diseases, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.
    Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context2021In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 413, p. 7157-7178Article, review/survey (Refereed)
    Abstract [en]

    The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.

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  • 11.
    Bally, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Department of Physics, Chalmers University of Technology, Gothenburg, Sweden.
    Pace, Hudson
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Höök, Fredrik
    Department of Physics, Chalmers University of Technology, Gothenburg, Sweden.
    Lipid-based bioanalytical sensors2021In: Handbook of lipid membranes: molecular, functional, and materials aspects / [ed] Cyrus R. Safinya; Joachim Rädler, CRC Press, 2021, p. 241-269Chapter in book (Refereed)
    Abstract [en]

    Lipid assemblies have attracted considerable interest as components in bioanalytical sensors. They provide a native-like environment for the immobilization of membrane proteins and for the study of membrane-related processes. Liposomes are also excellent bioanalytical assay components since selected functionalities can be added to the membrane while their aqueous interior can encapsulate a variety of molecules. This chapter highlights the potential of lipid assemblies in surface-based affinity sensors. It first describes how such sensors are created, providing an overview of lipid immobilization strategies together with a summary of the major transduction techniques used to probe binding at and transport through membrane interfaces. It then reviews the implementation of lipid-based sensors in the study of membrane proteins and membrane-mediated interactions, followed by a discussion of the potential of liposomes as nanoscale labels and as nanoreactors. Finally, it illustrates how external forces can be used to manipulate membrane component for biosensing applications.

  • 12.
    Bano, Fouzia
    et al.
    University of Liège, Department of Chemistry, Belgium.
    Sluysmans, D.
    Wislez, A.
    Duwez, A. -S
    Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy2015In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 7, no 46, p. 19528-19533Article in journal (Refereed)
    Abstract [en]

    Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold – a substrate commonly used in nanotechnology – and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA–gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide–Au bond strength. Force data show that, at physiological ionic strength, adenine–Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine–Au and guanine–Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.

  • 13.
    Bano, Fouzia
    et al.
    School of Biomedical Sciences, Faculty of Biological Sciences, School of Physics and Astronomy, Faculty of Mathematics and Physical Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom; CIC biomaGUNE, Biosurfaces Laboratory, Donostia-San Sebastian, Spain.
    Tammi, M. I.
    Kang, D. W.
    Harris, E. N.
    Richter, R. P.
    Single-molecule unbinding forces between the polysaccharide hyaluronan and its binding proteins2018In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 12, p. 2910-2922Article in journal (Refereed)
    Abstract [en]

    The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices.

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  • 14.
    Björnham, Oscar
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Catch-Bond behavior of bacteria binding by slip bonds2010In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 99, no 5, p. 1331-1341Article in journal (Refereed)
    Abstract [en]

    It is shown that multipili-adhering bacteria expressing helix-like pili binding by slip bonds can show catch-bond behavior. When exposed to an external force, such bacteria can mediate adhesion to their hosts by either of two limiting means: sequential or simultaneous pili force exposure (referring to when the pili mediate force in a sequential or simultaneous manner, respectively). As the force is increased, the pili can transition from sequential to simultaneous pili force exposure. Since the latter mode of adhesion gives rise to a significantly longer bacterial adhesion lifetime than the former, this results in a prolongation of the lifetime, which shows up as a catch-bond behavior. The properties and conditions of this effect were theoretically investigated and assessed in some detail for dual-pili-adhering bacteria, by both analytical means and simulations. The results indicate that the adhesion lifetime of such bacteria can be prolonged by more than an order of magnitude. This implies that the adhesion properties of multibinding systems cannot be directly conveyed to the individual adhesion-receptor bonds.

  • 15.
    Blanco-Rivero, Amaya
    et al.
    Madrid, Spain.
    Shutova, Tatiana
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    José Román, María
    Madrid, Spain.
    Villarejo, Arsenio
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain.
    Martinez, Flor
    Madrid, Spain.
    Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii2012In: PLOS ONE, E-ISSN 1932-6203, Vol. 7, no 11, article id e49063Article in journal (Refereed)
    Abstract [en]

    Background: Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO2 conditions. Results/Conclusions: In the present work we demonstrate that after transfer to low CO2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO2 conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. Significance: This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

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  • 16.
    Blomberg, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Höglund, Andreas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Eriksson, David
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Ruuth, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Jacobsson, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nilsson, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α in a resistant variant of the human U937 cell line2011In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 16, no 8, p. 783-794Article in journal (Refereed)
    Abstract [en]

    Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

  • 17. Chen, Mingzhi
    et al.
    Dousis, Athanasios D.
    Wuc, Yinghao
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ma, Jianpeng
    Predicting protein folding cores by empirical potential functions2009In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 483, no 1, p. 16-22Article in journal (Refereed)
    Abstract [en]

    Theoretical and in vitro experiments suggest that protein-folding cores form early in the process of folding, and that proteins may have evolved to optimize both folding speed and native-state stability. In our previous work (Chen et al., Structure, 14, 1401 (2006)), we developed a set of empirical potential functions and used them to analyze interaction energies among secondary-structure elements in two β-sandwich proteins. Our work on this group of proteins demonstrated that the predicted folding core also harbors residues that form native-like interactions early in the folding reaction. In the current work, we have tested our empirical potential functions on structurally-different proteins for which the folding cores have been revealed by protein hydrogen-deuterium exchange experiments. Using a set of 29 unrelated proteins, which have been extensively studied in the literature, we demonstrate that the average prediction result from our method is significantly better than predictions based on other computational methods. Our study is an important step towards the ultimate goal of understanding the correlation between folding cores and native structures.

  • 18. Chen, Xinyu
    et al.
    Yuan, Huwei
    Hu, Xiange
    Meng, Jingxiang
    Zhou, Xianqing
    Wang, Xiao-Ru
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. National Engineering Laboratory for Forest Tree Breeding, Key Laboratory of Genetic and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, People’s Republic of China.
    Li, Yue
    Variations in electrical impedance and phase angle among seedlings of Pinus densata and parental species in Pinus tabuliformis habitat environment2015In: Journal of Forestry Research, ISSN 1007-662X, E-ISSN 1993-0607, Vol. 26, no 3, p. 777-783Article in journal (Refereed)
    Abstract [en]

    Electrical impedance (EI) and phase angle (PHI) parameters in AC impedance spectroscopy are important electrical parameters in the study of medical pathology. However, little is known about their application in variation and genetic relationship studies of forest trees. In order to test whether impedance parameters could be used in genetic relationship analysis among conifer species, EI and PHI were measured in a seedling experiment test composed of Pinus tabuliformis, Pinus yunnanensis, and Pinus densata in a habitat of Pinus tabuliformis. The results showed that variations in both EI and PHI among species were significant in different electric frequencies, and the EI and PHI values measured in the two populations of P. densata were between the two parental species, P. yunnanensis and P. tabuliformis. These results show that these two impedance parameters could reflect the genetic relationship among pine species. This was the first time using the two AC impedance spectroscopy parameters to test the genetic relationship analysis between tree species, and would be a hopeful novel reference methodology for future studies in evolution and genetic variation of tree species.

  • 19.
    Cheregi, Otilia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Miranda, Hélder
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Inactivation of the Deg protease family in the cyanobacterium Synechocystis sp. PCC 6803 has impact on the outer cell layers2015In: Journal of Photochemistry and Photobiology. B: Biology, ISSN 1011-1344, E-ISSN 1873-2682, p. 383-394Article in journal (Refereed)
    Abstract [en]

    The serine type Deg/HtrA proteases are distributed in a wide range of organisms from Escherichia coli to humans. The cyanobacterium Synechocystis sp. PCC 6803 possesses three Deg protease orthologues: HtrA, HhoA and HhoB. Previously we compared Synechocystis 6803 wild type cells exposed to mild or severe stress conditions with a mutant lacking all three Deg proteases and demonstrated that stress had strong impact on the proteomes and metabolomes [1]. To identify the biochemical processes, which this protease family is involved in, here we compared Synechocystis sp. PCC 6803 wild type cells with a mutant lacking all three Deg proteases grown under normal growth conditions (30 °C and 40 μmol photons m−2 s−1). Deletion of the Deg proteases lead to the down-regulation of proteins related to the biosynthesis of outer cell layers (e.g. the GDP mannose 4,6-dehydratase) and affected protein secretion. During the late growth phase of the culture Deg proteases were found to be secreted to the extracellular medium of the Synechocystis sp. PCC 6803 wild type strain. While cyanobacterial Deg proteases seem to act mainly in the periplasmic space, deletion of the three proteases influences the proteome and metabolome of the whole cell. Impairments in the outer cell layers of the triple mutant might explain the higher sensitivity toward light and oxidative stress, which was observed earlier by Barker and coworkers [2].

  • 20.
    Christiansen, Alexander
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wang, Qian
    Department of Physics, University of Houston, Houston, TX, 77204, United States.
    Cheung, Margaret S.
    Department of Physics, University of Houston, Houston, TX, 77204, United States.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Effects of macromolecular crowding agents on protein folding in vitro and in silico2013In: Biophysical Reviews, ISSN 1867-2450, Vol. 5, no 2, p. 137-145Article, review/survey (Refereed)
    Abstract [en]

    Proteins fold and function inside cells which are environments very different from that of dilute buffer solutions most often used in traditional experiments. The crowded milieu results in excluded-volume effects, increased bulk viscosity and amplified chances for inter-molecular interactions. These environmental factors have not been accounted for in most mechanistic studies of protein folding executed during the last decades. The question thus arises as to how these effects-present when polypeptides normally fold in vivo-modulate protein biophysics. To address excluded volume effects, we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with proteins, in combination with strategically selected proteins and a range of equilibrium and time-resolved biophysical (spectroscopic and computational) methods. In this review, we describe key observations on macromolecular crowding effects on protein stability, folding and structure drawn from combined in vitro and in silico studies. As expected based on Minton's early predictions, many proteins (apoflavodoxin, VlsE, cytochrome c, and S16) became more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, unexpectedly, for apoflavodoxin and VlsE, the folded states changed both secondary structure content and, for VlsE, overall shape in the presence of macromolecular crowding. For apoflavodoxin and cytochrome c, which have complex kinetic folding mechanisms, excluded volume effects made the folding energy landscapes smoother (i. e., less misfolding and/or kinetic heterogeneity) than in buffer. 

  • 21.
    Christiansen, Alexander
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Quantification of excluded volume effects on the folding landscape of Pseudomonas aeruginosa Apoazurin In Vitro2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 105, no 7, p. 1689-1699Article in journal (Refereed)
    Abstract [en]

    Proteins fold and function inside cells that are crowded with macromolecules. Here, we address the role of the resulting excluded volume effects by in vitro spectroscopic studies of Pseudomonas aeruginosa apoazurin stability (thermal and chemical perturbations) and folding kinetics (chemical perturbation) as a function of increasing levels of crowding agents dextran (sizes 20, 40, and 70 kDa) and Ficoll 70. We find that excluded volume theory derived by Minton quantitatively captures the experimental effects when crowding agents are modeled as arrays of rods. This finding demonstrates that synthetic crowding agents are useful for studies of excluded volume effects. Moreover, thermal and chemical perturbations result in free energy effects by the presence of crowding agents that are identical, which shows that the unfolded state is energetically the same regardless of method of unfolding. This also underscores the two-state approximation for apoazurin’s unfolding reaction and suggests that thermal and chemical unfolding experiments can be used in an interchangeable way. Finally, we observe increased folding speed and invariant unfolding speed for apoazurin in the presence of macromolecular crowding agents, a result that points to unfolded-state perturbations. Although the absolute magnitude of excluded volume effects on apoazurin is only on the order of 1–3 kJ/mol, differences of this scale may be biologically significant.

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    Quantification of Excluded Volume Effects on the Folding Landscape of Pseudomonas aeruginosa Apoazurin In Vitro
  • 22.
    Clifton, Luke A.
    et al.
    ISIS Neutron and Muon Source, Science and Technology Facilities Council, Didcot, United Kingdom.
    Ul Mushtaq, Ameeq
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wacklin-Knecht, Hanna
    European Spallation Source, European Research Infrastructure Consortium, Lund, Sweden.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Insight into Bcl-2 proteins' functioning at mitochondrial membrane level2023In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 122, no 3S1, p. 232a-232a, article id 1130-PosArticle in journal (Refereed)
    Abstract [en]

    Programmed cell death (apoptosis) is essential in life. In its intrinsic apoptotic pathway opposing members of the B-cell lymphoma 2 (Bcl-2) protein family control the permeability of the mitochondrial outer membrane (MOM) and the release of apoptotic factors such as cytochrome c. Any misregulation of this process can cause disorders most prominently cancer, where often upregulation of cell protecting (anti-apoptotic) Bcl-2 members such as the Bcl-2 membrane protein itself plays a notorious role by blocking MOM perforation by - often drug induced - apoptotic proteins such as Bax which would cause cancer cell death normally. Here, we apply neutron reflectometry (NR) on supported lipid bilayers which mimic MOM environment and solid state/liquid state NMR spectroscopy to unravel the molecular basis driving opposing proteins to interact with each other at the MOM; a mechanism which is not really understood yet due to lack of high-resolution structural insight. Based on our central hypothesis that Bcl-2 drives its cell-protecting function at a membrane-embedded location as revealed by NR (1), we focus i) to determine the structure of human Bcl-2 protein in its membrane setting by combining solution and solid-state NMR; ii) use NR to study the kinetics and lipid/protein pore assemblied upon binding of Bax to mitochondrial membranes and its membrane destroying activities there; and iii) unravel the nature of direct interaction between Bcl-2 and Bax to neutralize each other. Knowledge generated here, will be indispensable in understanding the regulative function of the Bcl-2 family at mitochondrial membranes.

  • 23.
    Clifton, Luke A.
    et al.
    SIS Pulsed Neutron and Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot, Oxfordshire, UK.
    Wacklin-Knecht, Hanna P.
    European Spallation Source ERIC, ESS, Lund, Sweden; Department of Chemistry, Division of Physical Chemistry, Lund University, Lund, Sweden.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ul Mushtaq, Ameeq
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Creation of distinctive Bax-lipid complexes at mitochondrial membrane surfaces drives pore formation to initiate apoptosis2023In: Science Advances, E-ISSN 2375-2548, Vol. 9, no 22, article id eadg7940Article in journal (Refereed)
    Abstract [en]

    Apotosis is an essential process tightly regulated by the Bcl-2 protein family where proapoptotic Bax triggers cell death by perforating the mitochondrial outer membrane. Although intensively studied, the molecular mechanism by which these proteins create apoptotic pores remains elusive. Here, we show that Bax creates pores by extracting lipids from outer mitochondrial membrane mimics by formation of Bax/lipid clusters that are deposited on the membrane surface. Time-resolved neutron reflectometry and Fourier transform infrared spectroscopy revealed two kinetically distinct phases in the pore formation process, both of which were critically dependent on cardiolipin levels. The initially fast adsorption of Bax on the mitochondrial membrane surface is followed by a slower formation of pores and Bax-lipid clusters on the membrane surface. Our findings provide a robust molecular understanding of mitochondrial membrane perforation by cell-killing Bax protein and illuminate the initial phases of programmed cellular death. Bax initiates apoptosis by perforating mitochondrial membranes via formation of pores and extramembranous Bax-lipid complexes.

  • 24. da Hora, G. C. A.
    et al.
    Archilha, N. L.
    Lopes, J. L. S.
    Mueller, D. M.
    Coutinho, K.
    Itri, R.
    Soares, T. A.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Departamento de Quı´mica Fundamental, Universidade Federal de Pernambuco, 50740-560 Cidade Universita´ria, Recife, Brazil.
    Membrane negative curvature induced by a hybrid peptide from pediocin PA-1 and plantaricin 149 as revealed by atomistic molecular dynamics simulations2016In: Soft Matter, ISSN 1744-683X, E-ISSN 1744-6848, Vol. 12, no 43, p. 8884-8898Article in journal (Refereed)
    Abstract [en]

    Antimicrobial peptides (AMPs) are cationic peptides that kill bacteria with a broad spectrum of action, low toxicity to mammalian cells and exceptionally low rates of bacterial resistance. These features have led to considerable efforts in developing AMPs as an alternative antibacterial therapy. In vitro studies have shown that AMPs interfere with membrane bilayer integrity via several possible mechanisms, which are not entirely understood. We have performed the synthesis, membrane lysis measurements, and biophysical characterization of a novel hybrid peptide. These measurements show that PA-Pln149 does not form nanopores, but instead promotes membrane rupture. It causes fast rupture of the bacterial model membrane (POPG-rich) at concentrations 100-fold lower than that required for the disruption of mammalian model membranes (POPC-rich). Atomistic molecular dynamics (MD) simulations were performed for single and multiple copies of PA-Pln149 in the presence of mixed and pure POPC/POPG bilayers to investigate the concentration-dependent membrane disruption by the hybrid peptide. These simulations reproduced the experimental trend and provided a potential mechanism of action for PA-Pln149. It shows that the PA-Pln149 does not form nanopores, but instead promotes membrane destabilization through peptide aggregation and induction of membrane negative curvature with the collapse of the lamellar arrangement. The sequence of events depicted for PA-Pln149 may offer insights into the mechanism of action of AMPs previously shown to induce negative deformation of membrane curvature and often associated with peptide translocation via non-bilayer intermediate structures.

  • 25.
    Dahlberg, Tobias
    Umeå University, Faculty of Science and Technology, Department of Physics.
    KNOW YOUR ENEMY: Characterizing Pathogenic Biomaterials Using Laser Tweezers2022Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Diseases caused by pathogenic agents such as bacteria and viruses result in devastating costs on personal and societal levels. However, it is not just the emergence of new diseases that is problematic. Antibiotic resistance among bacteria makes uncomplicated infections difficult and lethal. Resilient disease-causing spores spread in hospitals, the food industry, and water supplies requiring effective detection and disinfection methods. Further, we face complex neurological diseases where no effective treatment or diagnostic methods exist. Thus, we must increase our fundamental understanding of these diseases to develop effective diagnostic, detection, disinfection, and treatment methods.

    Classically, the methods used for detecting and studying the underlying mechanics of pathogenic agents work on a large scale, measuring the average macroscopic behavior and properties of these pathogens. However, just as with humans, the average behavior is not always representative of individual behavior. Therefore, it is also essential to investigate the characteristics of these pathogens on a single cell or particle level. 

    This thesis develops and applies optical techniques to characterize pathogenic biomaterial on a single cell or particle level. At the heart of all these studies is our Optical Tweezers (OT) instrument. OT are a tool that allows us to reach into the microscopic world and interact with it. Finally, by combining OT with other experimental techniques, we can chemically characterize biomaterials and develop assays that mimic different biological settings. Using these tools, we investigate bacterial adhesion, disinfection, and detection of pathogenic spores and proteins.

    Hopefully, the insights of these studies can lessen the burden on society caused by diseases by helping others develop effective treatment, diagnostic, detection, and disinfection methods in the future. 

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  • 26.
    Dahlberg, Tobias
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Baker, Joseph
    Department of Chemistry, The College of New Jersey, Ewing, New Jersey.
    Bullitt, Esther
    Department of Physiology & Biophysics, Boston University School of Medicine, Boston, Massachusetts.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Unveiling molecular interactions that stabilize bacterial adhesion pili2022In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 121, no 11, p. 2096-2106Article in journal (Refereed)
    Abstract [en]

    Adhesion pili assembled by the chaperone-usher pathway are superelastic helical filaments on the surface of bacteria, optimized for attachment to target cells. Here, we investigate the biophysical function and structural interactions that stabilize P pili from uropathogenic bacteria. Using optical tweezers, we measure P pilus subunit-subunit interaction dynamics and show that pilus compliance is contour-length dependent. Atomic details of subunit-subunit interactions of pili under tension are shown using steered molecular dynamics (sMD) simulations. sMD results also indicate that the N-terminal “staple” region of P pili, which provides interactions with pilins that are four and five subunits away, significantly stabilizes the helical filament structure. These data are consistent with previous structural data, and suggest that more layer-to-layer interactions could compensate for the lack of a staple in type 1 pili. This study informs our understanding of essential structural and dynamic features of adhesion pili, supporting the hypothesis that the function of pili is critically dependent on their structure and biophysical properties.

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  • 27.
    Dahlberg, Tobias
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Baker, Joseph
    Dept of Chemistry, The College of New Jersey.
    Bullitt, Esther
    Dept of Physiology & Biophysics, Boston University School of Medicine.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Unveiling molecular interactions that stabilize the chaperone-usher pili rod and their role for mechanical and kinetic propertiesManuscript (preprint) (Other academic)
  • 28.
    Dahlberg, Tobias
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Malyshev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Per Ola
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Biophysical Fingerprinting of Single Bacterial Spores using Laser Raman Optical Tweezers2020In: Proceedings Volume 11416, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XXI, 2020, SPIE - International Society for Optical Engineering, 2020, article id 1141601Conference paper (Refereed)
    Abstract [en]

    Spore-forming bacteria that cause diseases pose a danger in our society. When in spore form, bacteria can survive high temperatures and resist a plethora of disinfection chemicals. Effective disinfection approaches are thus critical. Since a population of bacterial spores is heterogeneous in many aspects, single spore analyzing methods are suitable when heterogeneous information cannot be neglected. We present in this work a highresolution Laser Raman optical tweezers that can trap single spores and characterize their Raman spectra. We first evaluate our system by measuring Raman spectra of spores, and purified DNA and DPA. Thereafter, we expose Bacillus thuringiensis spores to peracetic acid, chlorine dioxide, and sodium hypochlorite, which are common disinfection chemicals. The data reveals how these agents change the constitutes of a spore over time, thus improving on the mode of action of these disinfection chemicals.

  • 29.
    de Lichtenberg, Casper
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Time-resolved Structural and Mechanistic Studies of Water Oxidation in Photosystem II: water here, water there, water everywhere2020Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Oxygenic photosynthesis is undisputedly one of the most important chemical processes for human life on earth as it not only fills the atmosphere with the oxygen that we need to breathe, but also sustains the accumulation of biomass, which is not only used as nourishment but is also present in almost every aspect of our lives as building material, textiles in clothes and furniture, or even as living decorations to name a few.

    The photosynthetic water-splitting mechanism is catalyzed by a water:plastoquinone oxido-reductase by the name of photosystem II (PSII), which is embedded in the thylakoid membranes of plants, algae and cyanobacteria. As it is excited by light, charge separation occurs in the reaction center of the protein and an electron is extracted by oxidation of Mn4Ca-cluster, that constitutes the active site for the water splitting reaction in PSII. When the Mn4Ca-cluster has been oxidized 4 times, it forms an oxygen-oxygen bond between two water derived ligands bound to the Mn4Ca-cluster and returns to the lowest oxidation state of the catalytic cycle. Understanding what ligands of the cluster that are used in the water splitting reaction is the key to unlocking the underlying chemical mechanism.

    In this thesis I describe investigations, with room temperature X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) on PSII microcrystals, of how the active site looks in all the stable intermediate oxidation states. Furthermore I describe how we uncovered the sequence of events that lead to insertion of an additional water ligand in the S2-S3 state transition of the catalytic cycle.

    Furthermore, through time-resolved membrane-inlet mass spectrometry (TR-MIMS) measurements of the isotopic equilibration of the substrate waters with the bulk in conditions that induce different electron magnetic resonance (EPR) spectroscopic signatures, I present evidence that the exchange of the slowly exchanging substrate water Ws is controlled by a dynamic equilibrium between conformations in the S2-state that give rise to either the low-spin multiline (LS-ML) signal or the high-spin (HS) signal. Based on the crystal structures and litterature suggestions for the conformation of the HS state different scenarios were presented for the assignment of Ws and how it exchanges. This analysis is discussed in the context of all semi-stable intermediate oxidation states in the Kok cycle.

    To further the understanding of this equilibrium, I also studied a selection of mutants positioned at strategic places in the vicinity of the different proposed substrates and at points that were suggested to be critical for substrate entry. With the combination of TR-MIMS and EPR, I reached the conclusion that by mutating valine 185 to asparagine, the water bound A-type conformation was stabilized, meanwhile in the mutant where aspartate 61 was mutated to alanine I observed that the barrier of the equilibrium between the exchanging conformations was so high that the interchange between them was arrested at room temperature. Additionally the retardation of the substrate exchange rates in the S3-states fit best with D61 being in the vicinity of the fast exchanging water. With this information we found the data best explained in a scenario where the water insertion of the S2-S3 transition was determining the if O-O bond formation occurred between the waters that were W2 and W3 or W2 and O5 in the S2 state. In addition, by mutation of glutamate 189 to glutamine that this residue is not important for the exchange of substrate waters in the S2 or the S3 states.

    Finally I use a combination of substrate labelling with TR-MIMS and time resolved labelling of the waters that ligate the Mn4Ca-cluster to show that the briding oxygen O5  is exchanging with a near identical rate to Ws, further supporting the assignment that Ws=O5.

    In conclusion, O-O bond formation most likely occurs between W2 (Wf) and O5 (Ws) via an oxo-oxyl radical coupling mechanism. The newly inserted water thus represents the slow exchanging water of the following S-state cycle.

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  • 30.
    de Lichtenberg, Casper
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Molecular Biomimetics, Department of Chemistry - Ångström, Uppsala University, Uppsala, Sweden.
    Avramov, Anton P.
    Zhang, Minquan
    Mamedov, Fikret
    Burnap, Robert L.
    Messinger, Johannes
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Molecular Biomimetics, Department of Chemistry - Ångström, Uppsala University, Uppsala, Sweden.
    The D1-V185N mutation alters substrate water exchange by stabilizing alternative structures of the Mn4Ca-cluster in photosystem II2021In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1862, no 1, article id 148319Article in journal (Refereed)
    Abstract [en]

    In photosynthesis, the oxygen-evolving complex (OEC) of the pigment-protein complex photosystem II (PSII) orchestrates the oxidation of water. Introduction of the V185N mutation into the D1 protein was previously reported to drastically slow O2-release and strongly perturb the water network surrounding the Mn4Ca cluster. Employing time-resolved membrane inlet mass spectrometry, we measured here the H218O/H216O-exchange kinetics of the fast (Wf) and slow (Ws) exchanging substrate waters bound in the S1, S2 and S3 states to the Mn4Ca cluster of PSII core complexes isolated from wild type and D1-V185N strains of Synechocystis sp. PCC 6803. We found that the rate of exchange for Ws was increased in the S1 and S2 states, while both Wf and Ws exchange rates were decreased in the S3 state. Additionally, we used EPR spectroscopy to characterize the Mn4Ca cluster and its interaction with the redox active D1-Tyr161 (YZ). In the S2 state, we observed a greatly diminished multiline signal in the V185N-PSII that could be recovered by addition of ammonia. The split signal in the S1 state was not affected, while the split signal in the S3 state was absent in the D1-V185N mutant. These findings are rationalized by the proposal that the N185 residue stabilizes the binding of an additional water-derived ligand at the Mn1 site of the Mn4Ca cluster via hydrogen bonding. Implications for the sites of substrate water binding are discussed.

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  • 31.
    de Lichtenberg, Casper
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kim, Christopher, J.
    Debus, Richard, J.
    Messinger, Johannes
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Effects of the D61A and E189Q mutations on the exchange of substrate water in photosystem IIManuscript (preprint) (Other academic)
  • 32.
    de Lichtenberg, Casper
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rapatskiy, Leonid
    Reus, Michael
    Heyno, Eiri
    Schnegg, Alexander
    Novaczyk, Marc, M.
    Lubitz, Wolfgang
    Messinger, Johannes
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cox, Nicholas
    Assignment of the slow exchanging substrate water of Nature's water splitting cofactorManuscript (preprint) (Other academic)
  • 33.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Josse, Pierre
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Dalinot, Clément
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Osmolovskyi, Artem
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Marqués, Pablo Simón
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Castán, José María Andrés
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Abad Galán, Laura
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Allain, Magali
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Khrouz, Lhoussain
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Maury, Olivier
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Le Bahers, Tangui
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Blanchard, Philippe
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Dabos-Seignon, Sylvie
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Monnereau, Cyrille
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cabanetos, Clément
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France; IRL CNRS 2002, 2BFUEL, CNRS -Yonsei University, Seoul, South Korea.
    Site-selected thionated benzothioxanthene chromophores as heavy-atom-free small-molecule photosensitizers for photodynamic therapy2022In: Communications Chemistry, E-ISSN 2399-3669, Vol. 5, article id 142Article in journal (Refereed)
    Abstract [en]

    Photodynamic therapy is a clinically approved anticancer modality that employs a light-activated agent (photosensitizer) to generate cytotoxic reactive oxygen species (ROS). There is therefore a growing interest for developing innovative photosensitizing agents with enhanced phototherapeutic performances. Herein, we report on a rational design synthetic procedure that converts the ultrabright benzothioxanthene imide (BTI) dye into three heavy-atom-free thionated compounds featuring close-to-unit singlet oxygen quantum yields. In contrast to the BTI, these thionated analogs display an almost fully quenched fluorescence emission, in agreement with the formation of highly populated triplet states. Indeed, the sequential thionation on the BTI scaffold induces torsion of its skeleton reducing the singlet-triplet energy gaps and enhancing the spin-orbit coupling. These potential PSs show potent cancer-cell ablation under light irradiation while remaining non-toxic under dark condition owing to a photo-cytotoxic mechanism that we believe simultaneously involves singlet oxygen and superoxide species, which could be both characterized in vitro. Our study demonstrates that this simple site-selected thionated platform is an effective strategy to convert conventional carbonyl-containing fluorophores into phototherapeutic agents for anticancer PDT.

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  • 34.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mosser, Maëlle
    Le Bahers, Tangui
    Dumont, Elise
    Dudek, Marta
    Denis-Quanquin, Sandrine
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Andraud, Chantal
    Matczyszyn, Katarzyna
    Monnereau, Cyrille
    Guy, Laure
    Light-induced in situ chemical activation of a fluorescent probe for monitoring intracellular G-quadruplex structures2021In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 13, no 32, p. 13795-13808Article in journal (Refereed)
    Abstract [en]

    Light-activated functional materials capable of remote control over duplex and G-quadruplex (G4) nucleic acids formation at the cellular level are still very rare. Herein, we report on the photoinduced macrocyclisation of a helicenoid quinoline derivative of binaphthol that selectively provides easy access to an unprecedented class of extended heteroaromatic structures with remarkable photophysical and DNA/RNA binding properties. Thus, while the native bisquinoline precursor shows no DNA binding activity, the new in situ photochemically generated probe features high association constants to DNA and RNA G4s. The latter inhibits DNA synthesis by selectively stabilizing G4 structures associated with oncogenic promoters and telomere repeat units. Finally, the light sensitive compound is capable of in cellulo photoconversion, localizes primarily in the G4-rich sites of cancer cells, competes with a well-known G4 binder and shows a clear nuclear co-localization with the quadruplex specific antibody BG4. This work provides a benchmark for the future design and development of a brand-new generation of light-activated target-selective G4-binders.

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  • 35.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Andréasson, Måns
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Tamilselvi, Shanmugam
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures2021In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 16, no 8, p. 1365-1376Article in journal (Refereed)
    Abstract [en]

    G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small “druglike” optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure–activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.

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  • 36.
    Dingeldein, Artur
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bax and oxidized phospholipids - a deadly complex: Apoptotic protein-lipid assemblies studied by MAS NMR spectroscopy2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Mitochondria are renowned for their vital role as cellular powerhouses because they provide ATP via cellular respiration. Additionally, these organelles also play an important role in other physiological processes, such as apoptosis. Apoptosis, or regulated cell death, is an important mechanism that regulates, for example, tissue homeostasis and embryonic development. Malfunctioning apoptosis can cause severe diseases such as various types of cancer and neurodegenerative diseases. The significance of mitochondria for apoptosis is that mitochondria host a variety of apoptogenic factors, such as cytochrome c. The release of these factors after the formation of an apoptotic pore can be regarded as a point of no return in the onset of apoptosis as these factors trigger the activation of caspases and consequently nuclear fragmentation.

    The mitochondrial outer membrane (MOM) is essential for deciding the cell’s fate, since the MOM provides an interaction surface for the pro- and anti-apoptotic proteins of the Bcl-2 protein family. Further, oxidized phospholipids (OxPls) within the MOM that are generated under oxidative stress conditions (a potent pro-apoptotic stimulus) can directly affect the equilibrium between pro- and anti-apoptotic proteins at the MOM surface, hence influencing the formation of apoptotic pores.

    To characterize the impact of different OxPls on membrane dynamics and organization, several MOM-mimicking systems were studied by solid-state magic angle spinning nuclear magnetic resonance spectroscopy (MAS NMR). These main experiments were accompanied by fluorescence spectroscopy and differential scanning calorimetry (DSC) studies to investigate the impact of OxPls and their interactions with the pro-apoptotic Bax protein at both the macroscopic and molecular levels. By combining several orthogonal methods, we were able to obtain a detailed description of the changes in MOM-mimicking vesicles induced by several types of OxPls. Moreover, we demonstrated how these changes impact the interaction between liposomes and the pro-apoptotic Bax protein.

    By using DSC, we were able to determine not only the macroscopic effect of two OxPls – PazePC and PoxnoPC – but also a concentration threshold. Both OxPls disrupted the membrane order such that the melting behavior of the MOM-mimicking vesicles became less cooperative. A decrease in cooperativity was detectable for OxPl concentrations of up to 5 mol%, after which the cooperativity remained constant. The addition of Bax resulted in an observable ordering effect, as some of the membrane disorder was negated by Bax and the melting process became more cooperative again. The ordering effect of Bax was subsequently confirmed by 31P MAS NMR experiments and cross polarization (CP) buildup curves. Analysis of the buildup curves revealed that at the molecular level, Bax enabled more efficient CP transfer, which indicated rigidification of the membranes after Bax insertion. Furthermore, the 31P NMR experiments provided the first molecular evidence of the importance of cardiolipin as a membrane contact site for Bax.

    Despite having similar disordering effects when studied with DSC, PoxnoPC and PazePC exhibited opposing effects on the pore formation ability of Bax. In studies with fluorescent dye-based leakage assays, Bax was able to form long-lived, stable pores in PazePC-containing giant unilamellar vesicles (GUVs). However, the observed leakage mechanism in PoxnoPC-containing GUVs could no longer be explained by an all-or-none leakage mechanism due to the brevity of the formed pores, leading to partially leaked vesicles, indicating a graded leakage mechanism instead.

    To investigate the possible reasons for the different leakage activities and to obtain mechanistic insights, we conducted 13C MAS NMR experiments. These experiments enabled us to pinpoint specific carbon sites in the different MOM-mimicking systems and to study their dynamic profile as a function of temperature. Though the OxPl-containing multilamellar vesicles (MLVs), compared with non-oxidized systems, also showed drastic dynamic changes, the molecular differences between PazePC- and PoxnoPC-containing vesicles were not significant enough to constitute a structural reason for the opposing leakage activities.

    In addition to investigating membrane dynamics, we were able to establish a novel strategy for producing cytotoxic Bax protein. This novel expression and purification strategy increased the obtained yields by an order of magnitude. By deploying a double fusion approach, we were able to inhibit both termini of the protein from exhibiting their disruptive, yield-diminishing interactions with the host cell membranes.

    In conclusion, over the course of this thesis we were able to develop fast, yet powerful tools to investigate the dynamic changes of MOM-mimicking vesicles under the influence of OxPls and pro-apoptotic Bax. In the future, these tools could be used to characterize the underlying protein-lipid interactions that are responsible for the opposing leakage activities. Due to the development of a novel Bax production strategy, future research could shift to protein-focused studies with the primary goal of determining the structure of the apoptotic Bax pore.

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  • 37.
    Dingeldein, Artur P G
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lidman, Martin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pokorna, Sarka
    Hof, Martin
    Pedersen, Anders
    Karlsson, Göran
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    BCL-2 Family Proteins Effect on Mitochondrial-Mimicking Membrane Structure by Solid State NMR2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 251A-252AArticle in journal (Other academic)
    Abstract [en]

    Mitochondria are not only the cells' powerhouse, but also involved in their suicide via apoptosis. Key regulators of this pathway are members of the Bcl-2 protein family which interact with the outer mitochondrial membrane to modulate permeability and enable the release of apoptotic stimuli like cytochrome c. For a long time the mitochondrial membrane forming lipids have been seen as merely structural building units with proteins doing the actual work. This view changed in recent years, since lipids were shown to be also directly involved in apoptotic events e.g. under intracellular oxidative stress. Oxidized phospholipids (OxPls) generated under these stress conditions might trigger mitochondria-mediated apoptosis. Their presence in mitochondrial membranes can severely alter the properties of these membranes with yet unknown consequences regarding the formation of pores through membrane-mediated interplay with apoptotic Bax protein. We therefore devised a model system that embodies oxidative stress conditions by incorporating OxPls into mitochondria mimicking model membranes composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) to study the impact of OxPls on apoptotic Bax-membrane interactions. To obtain molecular insight into hydrophobic fatty acid regions of membranes and their hydrophilic interface which is responsible for first protein-membrane contacts, we used differential scanning calorimetry (DSC) and solid state NMR spectroscopy. Upon incorporating OxPls with carboxyl (PoxnoPC) or aldehyde (PazePC) groups at their truncated sn-2-chains into our mitochondria model membranes, calorimetric and NMR measurements showed dramatic changes. 31P NMR experiments revealed major perturbation effects in these membranes; an effect which presumably elevates the membrane binding of apoptotic Bax to the charged membranes and its partial penetration, being a prerequisite for its final formation of pores which enable cytochrome c release from the mitochondrial interior. Currently structural studies of various Bax-lipid assemblies are ongoing.

  • 38.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lidman, Martin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dynamical and Structural Alterations withing Lipid-Protein Assemblies Control Apoptotic Pore Formation - A Solid State NMR Study2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 3, p. 59A-60AArticle in journal (Other academic)
  • 39.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Oxidatively stressed mitochondria-mimicking membranes: a molecular insight into their organization during apoptosis2018In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 12, p. 2644-2654Article in journal (Refereed)
    Abstract [en]

    Mitochondria are crucially involved in the removal of eukaryotic cells by the intrinsic pathway of programmed cell death (apoptosis). The mitochondrion's outer membrane (MOM) is the platform where this pathway takes place. Upon oxidative stress triggering apoptotic action, the MOM undergoes permeabilization and release of cytochrome c, ultimately causing cell death. This membrane perforation is regulated not only by opposing members of the Bcl-2 protein family meeting at the MOM but also actively the membrane itself. Upon oxidative damage, the membrane undergoes severe reorganization causing an increase in cell death-causing apoptotic Bcl-2 proteins. To understand the active role of MOM, we provided a detailed molecular view of its structural and dynamic reorganization upon oxidative stress by solid-state C-13 MAS NMR (magic angle spinning nuclear magnetic resonance) accompanied by calorimetric studies. By focusing on MOM-like vesicles doped with oxidized lipid species, direct polarization C-13 MAS NMR provided a quantitative overview and identification of all lipid moieties across the membrane. H-1-C-1(3) cross polarization and insensitive nuclei enhanced by polarization transfer MAS NMR generated a dynamic - mobile versus restricted - membrane profile. Oxidized phospholipids significantly perturb the structural membrane organization and increase membrane dynamics. These perturbations are not uniformly distributed as the hydrophobic core is reflecting the melting of lipid chains and increase in molecular disorder directly, whereas the interface and headgroup region undergo complex dynamical changes, reflecting increased intra-molecular flexibility of these moieties. These changes are potentially crucial in augmenting pro-apoptotic action of proteins like Bax.

  • 40.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wacklin, Hanna P.
    Clifton, Luke A.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mitochondrial Membrane Organization under Oxidative Stress: Insight by Solid-State NMR and Neutron Reflectometry2019In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, no 3, p. 508A-508AArticle in journal (Other academic)
  • 41.
    Dingeldein, Artur Peter Günther
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Åden, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wacklin, Hanna
    Sachl, Radek
    Pokorná, Sárka
    Hof, Martin
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Oxidatively Stressed Mitochondrial Membranes: Insight into their Organization and Function during Apoptosis2018Conference paper (Refereed)
  • 42.
    Domar, Ulla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Human intestinal alkaline phosphatase: tissue expression and serum levels1992Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Human alkaline phosphatase (ALP) comprises four isozymes, viz liver/bone/ kidney or tissue unspecific (AP), intestinal (LAP), placental (PLAP) and germ cell or PLAP-like alkaline phosphatase, with their main expression in specific tissues as indicated by their names. The isozymes are coded by different genes, but they are closely related, with more than 50% amino acid sequence homologies. Their biological function is unclear. In certain malignant and benign diseases, serum elevations of one or more of the isozymes occur, which is of diagnostic importance. In this study, the special expression of the intestinal isozyme in human tissues and sera, in normal as well as in pathological conditions, has been investigated by use of isozyme specific monoclonal antibodies.

    Monoclonal antibodies against the AP, IAP and PLAP isozymes were prepared, and specific assays developed, based on these monoclonal antibodies and the catalytic activity of the isozymes. By use of these assays the basal levels of all three isozymes were examined in selected normal organs. The isozymes were found to be expressed in measurable amounts in all the examined organs.

    IAP was immunohistochemically localized to the epithelial cells of membranes lining the ducts and tubules of the kidney, liver, pancreas and small intestine.

    Normal human serum contained all three isozymes. The AP isozyme constituted about 90% of the total ALP activity, the IAP isozyme less than 10% and the PLAP isozyme about 1%. Considerable interindividual variations of the serum IAP activity were observed. The serum activities of the IAP isozyme were related to the individual ABO blood group and secretor status. Non-secretors had low levels of IAP activity amounting to about one tenth of the activity in sera from blood group B or 0 secretors, while blood group A secretors had serum IAP activities in the same order as non-secretors. High individual day to day variations were observed.

    Fat absorption caused serum IAP to increase significantly for all persons, but it was rapidly cleared from the blood. We found that the release of IAP into the blood was linked to lipid absorption, but removal from the blood was not linked to lipoprotein clearance.

    Certain tumors of the testis expressed elevated levels of all three ALP isozymes. The highest activitiy of LAP was observed in one yolk sac tumor, in agreement with the endodermal origin of this tumor. In seminoma tissue the AP and PLAP isozymes were significantly, and IAP moderately elevated.

    Cirrhosis of the liver caused significantly increased serum levels of IAP besides the AP isozyme. In inflammatory diseases of the small intestine, normal serum IAP activities were observed.

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  • 43.
    Ehlers, Ina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    NMR studies of metabolites and xenobiotics: From time-points to long-term metabolic regulation2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Chemical species carry information in two dimensions, in their concentrations and their isotopic signatures. The concentrations of metabolites or synthetic compounds describe the composition of a chemical or biological system, while isotopic signatures describe processes in the system by their reaction pathways, regulation, and responses to external stimuli. Stable isotopes are unique tracers of these processes because their natural abundances are modulated by isotope effects occurring in physical processes as well as in chemical reactions. Nuclear magnetic resonance (NMR) spectroscopy is a prime technique not only for identification and quantification of small molecules in complex systems but also for measuring intramolecular distribution of stable isotopes in metabolites and other small molecules. In this thesis, we use quantitative NMR in three fields: in food science, environmental pollutant tracing, and plant-climate science.

    The phospholipid (PL) composition of food samples is of high interest because of their nutritional value and technological properties. However, the analysis of PLs is difficult as they constitute only a small fraction of the total lipid contents in foods. Here, we developed a method to identify PLs and determine their composition in food samples, by combining a liquid-liquid extraction approach for enriching PLs, with specialized 31P,1H-COSY NMR experiments to identify and quantify PLs.

    Wide-spread pollution with synthetic compounds threatens the environment and human health. However, the fate of pollutants in the environment is often poorly understood. Using quantitative deuterium NMR spectroscopy, we showed for the nitrosamine NDMA and the pesticide DDT how intramolecular distributions (isotopomer patterns) of the heavy hydrogen isotope deuterium reveal mechanistic insight into transformation pathways of pollutants and organic compounds in general. Intramolecular isotope distributions can be used to trace a pollutant’s origin, to understand its environmental transformation pathways and to evaluate remediation approaches.

    The atmospheric CO2 concentration ([CO2]) is currently rising at an unprecedented rate and plant responses to this increase in [CO2] influence the global carbon cycle and will determine future plant productivity. To investigate long-term plant responses, we developed a method to elucidate metabolic fluxes from intramolecular deuterium distributions of metabolites that can be extracted from historic plant material. We show that the intramolecular deuterium distribution of plant glucose depends on growth [CO2] and reflects the magnitude of photorespiration, an important side reaction of photosynthesis. In historic plant samples, we observe that photorespiration decreased in annual crop plants and natural vegetation over the past century, with no observable acclimation, implying that photosynthesis increased. In tree-ring samples from all continents covering the past 60 – 700 years, we detected a significantly smaller decrease in photorespiration than expected. We conclude that the expected “CO2 fertilization” has occurred but was significantly less pronounced in trees, due to opposing effects.

    The presented applications show that intramolecular isotope distributions not only provide information about the origin and turnover of compounds but also about metabolic regulation. By extracting isotope distributions from archives of plant material, metabolic information can be obtained retrospectively, which allows studies over decades to millennia, timescales that are inaccessible with manipulation experiments.

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  • 44.
    Eremenko, Ekaterina
    et al.
    Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
    Ben-Zvi, Anat
    National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Raveh, Dina
    Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
    Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model2013In: PLOS ONE, E-ISSN 1932-6203, Vol. 8, no 3, p. e58218-Article in journal (Refereed)
    Abstract [en]

    Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.

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  • 45. Figueroa Karlström, Eduardo
    et al.
    Lundström, Ronnie
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Occupational Medicine.
    Stensson, Olle
    Hansson Mild, Kjell
    Therapeutic staff exposure to magnetic field pulses during TMS/rTMS treatments2006In: Bioelectromagnetics, ISSN 0197-8462, E-ISSN 1521-186X, Vol. 27, no 2, p. 156-158Article in journal (Refereed)
    Abstract [en]

    Transcranial magnetic stimulation or repetitive transcranial magnetic stimulation (TMS/rTMS) is currently being used in treatments of the central nervous system diseases, for instance, depressive states. The principles of localized magnetic stimulation are summarized and the risk and level of occupational field exposure of the therapeutic staff is analyzed with reference to lCNIRP guidelines for pulses below 100 kHz. Measurements and analysis of the occupational exposure to magnetic fields of the staff working with TMS/rTMS are presented.

  • 46.
    Florea, Cristina
    et al.
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland.
    Tanska, Petri
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland.
    Mononen, Mika
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland.
    Qu, Chengjuan
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lammi, Mikko
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). School of Public Health, Health Science Center of Xi’an Jiaotong University, Key Laboratory of Trace Elements and Endemic Diseases, National Health and Family Planning Commission, Xi’an, China.
    Laasanen, Mikko
    School of Engineering and Technology, Savonia University of Applied Sciences, Kuopio, Finland.
    Korhonen, Rami
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland; Diagnostic Imaging Center, Kuopio University Hospital, Kuopio, Finland.
    A combined experimental atomic force microscopy-based nanoindentation and computational modeling approach to unravel the key contributors to the time-dependent mechanical behavior of single cells2017In: Biomechanics and Modeling in Mechanobiology, ISSN 1617-7959, E-ISSN 1617-7940, Vol. 16, no 1, p. 297-311, article id 27554263Article in journal (Refereed)
    Abstract [en]

    Cellular responses to mechanical stimuli are influenced by the mechanical properties of cells and the surrounding tissue matrix. Cells exhibit viscoelastic behavior in response to an applied stress. This has been attributed to fluid flow-dependent and flow-independent mechanisms. However, the particular mechanism that controls the local time-dependent behavior of cells is unknown. Here, a combined approach of experimental AFM nanoindentation with computational modeling is proposed, taking into account complex material behavior. Three constitutive models (porohyperelastic, viscohyperelastic, poroviscohyperelastic) in tandem with optimization algorithms were employed to capture the experimental stress relaxation data of chondrocytes at 5 % strain. The poroviscohyperelastic models with and without fluid flow allowed through the cell membrane provided excellent description of the experimental time-dependent cell responses (normalized mean squared error (NMSE) of 0.003 between the model and experiments). The viscohyperelastic model without fluid could not follow the entire experimental data that well (NMSE = 0.005), while the porohyperelastic model could not capture it at all (NMSE = 0.383). We also show by parametric analysis that the fluid flow has a small, but essential effect on the loading phase and short-term cell relaxation response, while the solid viscoelasticity controls the longer-term responses. We suggest that the local time-dependent cell mechanical response is determined by the combined effects of intrinsic viscoelasticity of the cytoskeleton and fluid flow redistribution in the cells, although the contribution of fluid flow is smaller when using a nanosized probe and moderate indentation rate. The present approach provides new insights into viscoelastic responses of chondrocytes, important for further understanding cell mechanobiological mechanisms in health and disease.

  • 47.
    Freire, Rafael V.M.
    et al.
    Department of Chemistry, University of Fribourg, Chemin Du Musée 9, 1700 Fribourg, Switzerland.
    Pillco-Valencia, Yeny
    Department of Fundamental Chemistry, Federal University of Pernambuco, Cidade Universitária, 50740-560 Recife, Brazil.
    da Hora, Gabriel C.A.
    Department of Fundamental Chemistry, Federal University of Pernambuco, Cidade Universitária, 50740-560 Recife, Brazil.
    Ramstedt, Madeleine
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sandblad, Linda
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Soares, Thereza A.
    Department of Fundamental Chemistry, Federal University of Pernambuco, Cidade Universitária, 50740-560 Recife, Brazil.
    Salentinig, Stefan
    epartment of Chemistry, University of Fribourg, Chemin Du Musée 9, 1700 Fribourg, Switzerland.
    Antimicrobial peptide induced colloidal transformations in bacteria-mimetic vesicles: combining in silico tools and experimental methods2021In: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 596, p. 352-363Article in journal (Refereed)
    Abstract [en]

    With the growing challenges of bacteria becoming resistant to conventional antibiotics, antimicrobial peptides (AMPs) may offer a potential alternative. One of the most studied AMPs, the human cathelicidin derived AMP LL-37 is notable for its antimicrobial activity even though its mechanism of action is not fully understood yet. This work investigates the interaction of LL-37 with 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG) vesicles, which were employed as a bacterial membrane model given the common presence of this phospholipid in the bacterial membrane. Experimental techniques including small angle X-ray scattering, transmission electron microscopy and dynamic light scattering were used to characterize the interactions among LL-37 and POPG. Molecular dynamics simulations complement the experimental studies with molecular-level insights into the process. LL-37 was discovered to actively and critically interact with the POPG vesicles, modifying the membrane curvature that eventually leads to structural transformations from vesicles to mixed micelles. The results shed light on the mechanisms underlying the interactions among LL-37 and bacteria mimetic vesicles and can guide the further development of AMP based antimicrobial materials and therapies.

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  • 48. Frost, Rickard
    et al.
    Débarre, Delphine
    Jana, Saikat
    Bano, Fouzia
    School of Biomedical Sciences, Faculty ofBiological Sciences, School of Physics and Astronomy, Faculty ofEngineering and Physical Sciences, Astbury Centre of StructuralMolecular Biology, and Bragg Centre for Materials Research,University of Leeds, Leeds LS2 9JT, United Kingdom.
    Schünemann, Jürgen
    Görlich, Dirk
    Richter, Ralf P.
    A method to quantify molecular diffusion within thin solvated polymer films: A case study on films of natively unfolded nucleoporins2020In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 14, no 8, p. 9938-9952Article in journal (Refereed)
    Abstract [en]

    We present a method to probe molecular and nanoparticle diffusion within thin, solvated polymer coatings. The device exploits the confinement with well-defined geometry that forms at the interface between a planar and a hemispherical surface (of which at least one is coated with polymers) in close contact and uses this confinement to analyze diffusion processes without interference of exchange with and diffusion in the bulk solution. With this method, which we call plane–sphere confinement microscopy (PSCM), information regarding the partitioning of molecules between the polymer coating and the bulk liquid is also obtained. Thanks to the shape of the confined geometry, diffusion and partitioning can be mapped as a function of compression and concentration of the coating in a single experiment. The method is versatile and can be integrated with conventional optical microscopes; thus it should find widespread use in the many application areas exploiting functional polymer coatings. We demonstrate the use of PSCM using brushes of natively unfolded nucleoporin domains rich in phenylalanine–glycine repeats (FG domains). A meshwork of FG domains is known to be responsible for the selective transport of nuclear transport receptors (NTRs) and their macromolecular cargos across the nuclear envelope that separates the cytosol and the nucleus of living cells. We find that the selectivity of NTR uptake by FG domain films depends sensitively on FG domain concentration and that the interaction of NTRs with FG domains obstructs NTR movement only moderately. These observations contribute important information to better understand the mechanisms of selective NTR transport.

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  • 49.
    Fällman, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Jass, Jana
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion2004In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 19, no 11, p. 1429-1437Article in journal (Refereed)
    Abstract [en]

    An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle’s displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads

  • 50.
    Giatrellis, Sarantis
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nounesis, George
    Biomolecular Physics Laboratory, IRRP, National Centre for Scientific Research Demokritos, 153 10 Aghia Paraskevi, Greece.
    Nucleic acid-lipid membrane interactions studied by DSC2011In: Journal of Pharmacy and Bioallied Sciences, ISSN 0975-7406, Vol. 3, no 1, p. 70-76Article in journal (Refereed)
    Abstract [en]

    The interactions of nucleic acids with lipid membranes are of great importance for biological mechanisms as well as for biotechnological applications in gene delivery and drug carriers. The optimization of liposomal vectors for clinical use is absolutely dependent upon the formation mechanisms, the morphology, and the molecular organization of the lipoplexes, that is, the complexes of lipid membranes with DNA. Differential scanning calorimetry (DSC) has emerged as an efficient and relatively easy-to-operate experimental technique that can straightforwardly provide data related to the thermodynamics and the kinetics of the DNA-lipid complexation and especially to the lipid organization and phase transitions within the membrane. In this review, we summarize DSC studies considering nucleic acid-membrane systems, accentuating DSC capabilities, and data analysis. Published work involving cationic, anionic, and zwitterionic lipids as well as lipid mixtures interacting with RNA and DNA of different sizes and conformations are included. It is shown that despite limitations, issues such as DNA- or RNA-induced phase separation and microdomain lipid segregation, liposomal aggregation and fusion, alterations of the lipid long-range molecular order, as well as membrane-induced structural changes of the nucleic acids can be efficiently treated by systematic high-sensitivity DSC studies.

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