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  • 1.
    Aguilar, Ximena
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Macromolecular crowding extended to a heptameric system: the co-chaperonin protein 102011In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 14, 3034-3044 p.Article in journal (Refereed)
    Abstract [en]

    Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.

  • 2.
    Bavdek, Andrej
    et al.
    Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Gekara, Nelson O
    Priselac, Dragan
    Gutiérrez Aguirre, Ion
    Darji, Ayub
    Chakraborty, Trinad
    Macek, Peter
    Lakey, Jeremy H
    Weiss, Siegfried
    Anderluh, Gregor
    Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O.2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 14, 4425-4437 p.Article in journal (Refereed)
    Abstract [en]

    Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3beta-hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.

  • 3.
    Björklund, Stefan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Skog, Sven
    Tribukait, Bernard
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    S-phase-specific expression of mammalian ribonucleotide reductase R1 and R2 subunit mRNAs1990In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 29, no 23, 5452-5458 p.Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein is present in excess in proliferating cells, and its levels are constant during the cell cycle. Expression of the R2 protein, which is limiting for enzyme activity, is strictly S-phase-correlated. In this paper, we have used antisense RNA probes in a solution hybridization assay to measure the levels of R1 and R2 mRNA during the cell cycle in centrifugally elutriated cells and in cells synchronized by isoleucine or serum starvation. The levels of both transcripts were very low or undetectable in G0/G1-phase cells, showed a pronounced increase as cells progressed into S phase, and then declined when cells progressed into G2 + M phase. The R1 and R2 transcripts increased in parallel, starting slightly before the rise in S-phase cells, and reached the same levels. The relative lack of cell cycle dependent variation in R1 protein levels, obtained previously, may therefore simply be a consequence of the long half-life of the R1 protein. Hydroxyurea-resistant, R2-overproducing mouse TA3 cells showed the same regulation of the R1 and R2 transcripts as the parental cells, but with R2 mRNA at a 40-fold higher level.

  • 4.
    Chen, Eefei
    et al.
    Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
    Christiansen, Alexander
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wang, Qian
    Department of Physics, University of Houston, Houston, Texas 77204, United States.
    Cheung, Margaret S
    Department of Physics, University of Houston, Houston, Texas 77204, United States.
    Kliger, David S
    Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 49, 9836-9845 p.Article in journal (Refereed)
    Abstract [en]

    Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.

  • 5.
    Christiansen, Alexander
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wang, Qian
    Department of Physics, University of Houston, Houston, Texas, USA .
    Samiotakis, Antonios
    Department of Physics, University of Houston, Houston, Texas, USA .
    Cheung, Margaret S
    Department of Physics, University of Houston, Houston, Texas, USA .
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Factors defining effects of Macromolecular crowding on protein stability: an in vitro/in silico case study using cytochrome c2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 31, 6519-6530 p.Article in journal (Refereed)
    Abstract [en]

    Previous experiments with two single-domain proteins showed that macromolecular crowding can stabilize dramatically toward heat perturbation and modulate native-state structure and shape. To assess the generality of this, we here tested the effects of the synthetic crowding agents on cytochrome c, a small single-domain protein. Using far-UV circular dichroism (CD), we discovered that there is no effect on cytochrome c's secondary structure upon addition of Ficoll or dextran (0-400 mg/mL, pH 7). Thermal experiments revealed stabilizing effects (5-10 degrees C) of Ficoll 70 and dextran 70; this effect was enhanced by the presence of low levels of guanidine hydrochloride (GuHCl) that destabilize the protein. When using a smaller dextran, dextran 40, the thermal effects were larger (10-20 degrees C). In silico analysis, using structure-based (Go-like) interactions for cytochrome c, is in excellent agreement with the in vitro thermodynamic data and also agrees with scaled particle theory. Simulations of a range of crowder size and shape demonstrated that the smaller the crowder the larger the favorable effect on cytochrome c's folded-state stability. Together with previous data, we conclude that protein size, stability, conformational malleability, and folding routes, as well as crowder size and shape, are key factors that modulate the net effect of macromolecular crowding on proteins.

  • 6. Fa, M
    et al.
    Karolin, J
    Aleshkov, S
    Strandberg, L
    Johansson, L B
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin.1995In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 34, no 42, 13833-40 p.Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)

  • 7. Gavriljuk, Konstantin
    et al.
    Schartner, Jonas
    Seidel, Hans
    Dickhut, Clarissa
    Zahedi, Rene P.
    Hedberg, Christian
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Kötting, Carsten
    Gerwert, Klaus
    Unraveling the Phosphocholination Mechanism of the Legionella pneumophila Enzyme AnkX2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 31, 4375-4385 p.Article in journal (Refereed)
    Abstract [en]

    The intracellular pathogen Legionella pneumophila infects lung macrophages and injects numerous effector proteins into the host cell to establish a vacuole for proliferation. The necessary interference with vesicular trafficking of the host is achieved by modulation of the function of Rab GTPases. The effector protein AnkX chemically modifies Rab1b and Rab35 by covalent phosphocholination of serine or threonine residues using CDP-choline as a donor. So far, the phosphoryl transfer mechanism and the relevance of observed autophosphocholination of AnkX remained disputable. We designed tailored caged compounds to make this type of enzymatic reaction accessible for time-resolved Fourier transform infrared difference spectroscopy. By combining spectroscopic and biochemical methods, we determined that full length AnkX is autophosphocholinated at Ser521, Thr620, and Thr943. However, autophosphocholination loses specificity for these sites in shortened constructs and does not appear to be relevant for the catalysis of the phosphoryl transfer. In contrast, transient phosphocholination of His229 in the conserved catalytic motif might exist as a short-lived reaction intermediate. Upon substrate binding, His229 is deprotonated and locked in this state, being rendered capable of a nucleophilic attack on the pyrophosphate moiety of the substrate. The proton that originated from His229 is transferred to a nearby carboxylic acid residue. Thus, our combined findings support a ping-pong mechanism involving phosphocholination of His229 and subsequent transfer of phosphocholine to the Rab GTPase. Our approach can be extended to the investigation of further nucleotidyl transfer reactions, which are currently of reemerging interest in regulatory pathways of host–pathogen interactions.

  • 8.
    Goldsteins, Gundars
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Andersson, Karin
    Umeå University, Faculty of Medicine, Molecular Biology.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Molecular Biology.
    Dacklin, Ingrid
    Umeå University, Faculty of Medicine, Molecular Biology.
    Edvinsson, Åsa
    Umeå University, Faculty of Medicine, Molecular Biology.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Sandgren, Ola
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Thylén, Christina
    Umeå University, Faculty of Medicine, Molecular Biology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Molecular Biology.
    Characterisation of two highly amyloidogenic mutants of transthyretin.1997In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 18, 5346-5352 p.Article in journal (Refereed)
    Abstract [en]

    The plasma protein transthyretin (TTR) has the potential to form amyloid under certain conditions. More than 50 different point mutations have been associated with amyloid formation that occurs only in adults. It is not known what structural changes are introduced into the structure of this otherwise stable molecule that results in its aggregation into insoluble amyloid fibrils. On the basis of calculations of the frequency of known mutations over the polypeptide, we have constructed two mutants in the D-strand of the polypeptide. These molecules, containing either a deletion or a substitution at amino acid positions 53−55, were unstable and spontaneously formed aggregates upon storage in TBS (pH 7.6). The precipitates were shown to be amyloid by staining with thioflavin T and Congo Red. Their ultrastructure was very similar to that of amyloid fibrils deposited in the vitreous body of patients with familial amyloidotic polyneuropathy type 1 with an amino acid replacement in position 30 (TTRmet30). Like amyloid isolated from the vitreous body of the eye, the amyloid precipitates generated from the TTR mutants exposed a trypsin cleavage site between amino acid residues 48 and 49, while plasma TTRmet30 isolated from amyloidosis patients as well as wild-type TTR only showed minor trypsin sensitivity. Our data indicate that the mutants we have constructed are similar to amyloid precursors or may share structural properties with intermediates on a pathway leading to amyloid deposits of plasma TTR.

  • 9.
    Hörnberg, Andreas
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Wikström Hultdin, Ulrika
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The effect of iodide and chloride on transthyretin structure and stability2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 26, 9290-9299 p.Article in journal (Other academic)
    Abstract [en]

    Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.

  • 10.
    Karlsson, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Eneqvist, Therese
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Cys114-linked dimers of transthyretin are compatible with amyloid formation.2005In: Biochemistry, ISSN 0006-2960, Vol. 44, no 39, 13063-70 p.Article in journal (Refereed)
    Abstract [en]

    The Tyr114Cys substitution in the human plasma protein transthyretin leads to a particularly aggressive form of familial amyloidotic polyneuropathy. In a previous study we demonstrated that ATTR Tyr114Cys forms intermolecular disulfide bonds, which partly impair fibril formation and result in a more amorphous morphology. Apart from the introduced cysteinyl group in position 114, the native sequence contains one cysteine located at position 10. To deduce the role of intermolecular disulfide bridging in fibril formation we generated and characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular cysteine bridge at position 114 enhances the exposure of cysteine 10, thereby facilitating additional intermolecular cysteine assemblies. We also purified a disulfide-linked dimeric form of TTR Cys10Ala/Tyr114Cys, which was recognized by the anti-TTR amyloid-specific monoclonal antibody MAb (39-44). Moreover, this dimeric molecule can form protofibrils indistinguishable from the fibrils formed under reducing conditions, as judged by atomic force microscopy. Assuming that both molecules of the dimer are part of the core of the fibril, the assembly is incompatible with a preserved native or near-native dimeric interphase. Our findings raise the question of whether TTR-amyloid architecture is indeed the result of one highly stringent assembly of structures or if different fibrils may be built from different underlying structures.

  • 11.
    Morozova-Roche, Ludmilla
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zamotin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Malisauskas, Mantas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chertkova, Rita
    Lavrikova, Marika A
    Kostanyan, Irina A
    Dolgikh, Dmiltry A
    Kirpichnikov, Michail P
    Fibrillation of Carrier Protein Albebetin and Its Biologically Active Constructs. Multiple Oligomeric Intermediates and Pathways2004In: Biochemistry, ISSN 0006-2960, Biochemistry, Vol. 43, no 30, 9610-9619 p.Article in journal (Refereed)
    Abstract [en]

    We showed that the genetically engineered carrier-protein albebetin and its biologically active constructs with interferon-2 octapeptide LKEKKYSP or differentiation factor hexapeptide TGENHR are inherently highly amyloidogenic at physiological pH. The kinetics of fibrillation were monitored by thioflavine-T (ThT) binding and the morphological changes by atomic force microscopy. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils. Comparative height and volume microscopic measurements allowed us to identify two distinct types of oligomeric intermediates: pivotal oligomers ca. 1.2 nm in height comprised of 10-12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height constituted of 26-30 molecules. The former assemble into chains and rings with "bead-on-string morphology", in which a "bead" corresponds to an individual oligomer. Once formed, the rings and chains remain in solution simultaneously with fibrils. The latter give rise to protofilaments and fibrils, and their formation is concomitant with an increasing level of ThT binding. The amyloid nature of filamentous structures was confirmed by a pronounced ThT and Congo red binding and -sheet-rich far-UV circular dichroism. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either fused to albebetin or added into solution, and an increased ionic strength promote fibrillation of albebetin (net charge of -12) by counterbalancing critical electrostatic repulsions. This finding demonstrates that the fibrillation of newly designed polypeptide-based products can produce multimeric amyloid species with a potentially "new" functionality, raising questions about their safety.

  • 12.
    Nilsson, Stefan K
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Lookene, Aivar
    Beckstead, Jennifer A
    Gliemann, Jørgen
    Ryan, Robert O
    Olivecrona, Gunilla
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Apolipoprotein A-V interaction with members of the low density lipoprotein receptor gene family2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 12, 3896-3904 p.Article in journal (Refereed)
  • 13.
    Onions, Jacqueline
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Hermann, Stefan
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    A novel type of calmodulin interaction in the inhibition of basic helix-loop-helix transcription factors.2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 15, 4366-4374 p.Article in journal (Refereed)
    Abstract [en]

    Calmodulin is the predominant intracellular receptor for Ca(2+) signals, mediating the regulation of numerous cellular processes. Previous studies have shown that calcium-loaded calmodulin can bind to and inhibit the activity of certain basic helix-loop-helix (bHLH) transcription factors. The basic sequence within the bHLH domain is the primary target for calmodulin binding, and sequences modulating the calmodulin interaction reside directly N-terminal to the basic sequence. Here we show that the interaction of calmodulin with bHLH proteins is of a novel type, displaying characteristics very different from those of previously characterized calmodulin-target complexes. We show that calmodulin interacts much stronger with a dimeric basic sequence than with the monomeric form. The calmodulin-bHLH protein complex contains equimolar amounts of calmodulin and bHLH chains. The interaction is unusual in being to a large extent polar in nature, and it is highly resistant to tested calmodulin inhibitors. Both the N-terminal and C-terminal domains of calmodulin can independently bind to and inhibit the DNA binding of bHLH proteins. The C-terminal domain preferentially binds to the basic sequence, whereas the N-terminal domain is essential for the effect of the modulatory sequence. We propose a model for the calmodulin-bHLH complex where two calmodulin molecules interact with one bHLH dimer, with one domain of calmodulin preferentially binding to the basic sequence of bHLH proteins and the other domain interacting with the modulatory sequence.

  • 14. Popović-Bijelić, Ana
    et al.
    Voevodskaya, Nina
    Domkin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gräslund, Astrid
    Metal binding and activity of ribonucleotide reductase protein R2 mutants: conditions for formation of the mixed manganese-iron cofactor2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 27, 6532-6539 p.Article in journal (Refereed)
    Abstract [en]

    Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.

  • 15. Qian, H
    et al.
    Edlund, U
    Powlowski, J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sethson, I
    Solution structure of phenol hydroxylase protein component P2 determined by NMR spectroscopy.1997In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 3Article in journal (Refereed)
    Abstract [en]

    Phenol hydroxylase from Pseudomonas sp. CF600 is a member of a family of binuclear iron-center-containing multicomponent oxygenases, which catalyzes the conversion of phenol and some of its methyl-substituted derivatives to catechol. In addition to a reductase component which transfers electrons from NADH, optimal turnover of the hydroxylase requires P2, a protein containing 90 amino acids which is readily resolved from the other components. The three-dimensional solution structure of P2 has been solved by 3D heteronuclear NMR spectroscopy. On the basis of 1206 experimental constraints, including 1060 distance constraints obtained from NOEs, 70 phi dihedral angle constraints, 42 psi dihedral angle constraints, and 34 hydrogen bond constraints, a total of 12 converged structures were obtained. The atomic root mean square deviation for the 12 converged structure with respect to the mean coordinates is 2.48 A for the backbone atoms and 3.85 A for all the heavy atoms. This relatively large uncertainty can be ascribed to conformational flexibility and exchange. The molecular structure of P2 is composed of three helices, six antiparallel beta-strands, one beta-hairpin, and some less ordered regions. This is the first structure among the known multicomponent oxygenases. On the basis of the three-dimensional structure of P2, sequence comparisons with similar proteins from other multicomponent oxygenases suggested that all of these proteins may have a conserved structure in the core regions.

  • 16.
    Rundqvist, Louise
    et al.
    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.
    Wallgren, Marcus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olsson, Ulrika
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Noncooperative folding of subdomains in Adenylate Kinase2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 9, 1911-1927 p.Article in journal (Refereed)
    Abstract [en]

    Conformational change is regulating the biological activity of a large number of proteins and enzymes. Efforts in structural biology have provided molecular descriptions of the interactions that stabilize the stable ground states on the reaction trajectories during conformational change. Less is known about equilibrium thermodynamic stabilities of the polypeptide segments that participate in structural changes and whether the stabilities are relevant for the reaction pathway. Adenylate kinase (Adk) is composed of three subdomains: CORE, ATPlid, and AMPbd. ATPlid and AMPbd are flexible nucleotide binding subdomains where large-scale conformational changes are directly coupled to catalytic activity. In this report, the equilibrium thermodynamic stabilities of Adk from both mesophilic and hyperthermophilic bacteria were investigated using solution state NMR spectroscopy together with protein engineering experiments. Equilibrium hydrogen to deuterium exchange experiments indicate that the flexible subdomains are of significantly lower thermodynamic stability compared to the CORE subdomain. Using site-directed mutagenesis, parts of ATPlid and AMPbd could be selectively unfolded as a result of perturbation of hydrophobic clusters located in these respective subdomains. Analysis of the perturbed Adk variants using NMR spin relaxation and Cα chemical shifts shows that the CORE subdomain can fold independently of ATPlid and AMPbd; consequently, folding of the two flexible subdomains occurs independently of each other. Based on the experimental results it is apparent that the flexible subdomains fold into their native structure in a noncooperative manner with respect to the CORE subdomain. These results are discussed in light of the catalytically relevant conformational change of ATPlid and AMPbd.

  • 17.
    Shutova, Tatyana
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Nikitina, Julia
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Deikus, Gintaras
    Andersson, Bertil
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Klimov, Vyacheslav
    Samuelsson, Göran
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Structural dynamics of the manganese-stabilizing protein-effect of pH, calcium, and manganese2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 46, 15182-15192 p.Article in journal (Refereed)
    Abstract [en]

    The photosystem-II-associated 33-kDa extrinsic manganese-stabilizing protein is found in all oxygen-evolving organisms. In this paper, we show that this protein undergoes pH-induced conformational changes in the physiological pH range. At a neutral pH of 7.2, the hydrophobic amino acid residues that are most likely located inside the barrel are "closed" and the protein binds neither Mn2+ nor Ca2+ ions. When the protein is transferred to a solution with a slightly acidic pH of 5.7, hydrophobic amino acid residues become exposed to the surrounding medium, enabling them to bind the fluorescent probe 8,1-ANS. At this pH-induced open state, Mn2+ and Ca2+ bind to the manganese-stabilizing protein. The pH values used in this study, 7.2 and 5.7, are typical of the pH found in the thylakoid lumen in the dark and light, respectively. A model is presented in which the manganese-stabilizing protein undergoes a pH-dependent conformational change that in turn influences its capacity to bind calcium and manganese. In this model, the proton-dependent conformational changes of the tertiary structure of the manganese-stabilizing protein are of functional relevance for the regulation of substrate (water) delivery to and product (proton) release from the water-oxidizing complex by forming a proton-sensing proton-transport pathway.

  • 18. Trelle, Morten Beck
    et al.
    Hirschberg, Daniel
    Jansson, Anna
    Ploug, Michael
    Roepstorff, Peter
    Andreasen, Peter A
    Jørgensen, Thomas J D
    Hydrogen/deuterium exchange mass spectrometry reveals specific changes in the local flexibility of plasminogen activator inhibitor 1 upon binding to the somatomedin B domain of vitronectin.2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 41Article in journal (Refereed)
    Abstract [en]

    The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1.

  • 19. Van Dael, Herman
    et al.
    Haezebrouck, Petra
    Morozova, Ludmilla
    Max Planck Institute fur Biochemie, Martinsried, Germany.
    Arico-Muendel, Christopher
    Dobson, Christopher M
    Partially folded states of equine lysozyme. Structural characterization and significance for protein folding.1993In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 32, no 44, 11886-11894 p.Article in journal (Refereed)
    Abstract [en]

    Despite their homologous structure, c-type lysozymes and alpha-lactalbumins have been found to differ profoundly in their unfolding behavior, in that the alpha-lactalbumins readily enter a partially unfolded collapsed state (the "molten globule"), whereas lysozymes unfold cooperatively to a highly unfolded state. The calcium-binding property of lysozyme from equine milk provides an evolutionary link between the two families of proteins. We demonstrate here that equine lysozyme undergoes a two-stage unfolding transition upon heating or in the presence of guanidine hydrochloride that is highly dependent on the state of calcium binding. Differential scanning calorimetry shows the two transitions to be particularly well resolved in the calcium-free protein, where the first transition occurs with a midpoint at 44 degrees C at pH 4.5 or in 0.8 M GdnHCl at pH 7.5, 25 degrees C, and the second occurs near 70 degrees C at pH 4.5 or in 3.7 M GdnHCl at pH 7.5, 25 degrees C. In the presence of calcium, the first transition takes place with a midpoint of 55 degrees C or in excess of 2.5 M GdnHCl, but the parameters for the second transition remain unchanged. Fluorescence emission and UV difference absorption spectroscopy suggest that the first transition generates an intermediate state in which sequestration of some aromatic side chains from solvent has occurred whereas the second represents denaturation to a highly unfolded state. CD and 1H NMR results indicate that the intermediate state possesses extensive secondary and tertiary structure, although the latter is substantially disordered.(ABSTRACT TRUNCATED AT 250 WORDS)

  • 20. Villebeck, Laila
    et al.
    Persson, Malin
    Luan, Shi-Lu
    Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Biomedical Laboratory Science.
    Hammarström, Per
    Lindgren, Mikael
    Jonsson, Bengt-Harald
    Conformational rearrangements of tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC)-bound actin.2007In: Biochemistry, ISSN 0006-2960, Vol. 46, no 17, 5083-93 p.Article in journal (Refereed)
    Abstract [en]

    The mechanism of chaperonins is still under intense investigation. Earlier studies by others and us on the bacterial chaperonin GroEL points to an active role of chaperonins in unfolding the target protein during initial binding. Here, a natural eukaryotic chaperonin system [tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC) and its target protein actin] was investigated to determine if the active participation of the chaperonin in the folding process is evolutionary-conserved. Using fluorescence resonance energy transfer (FRET) measurements on four distinct doubly fluorescein-labeled variants of actin, we have obtained a fairly detailed map of the structural rearrangements that occur during the TRiC-actin interaction. The results clearly show that TRiC has an active role in rearranging the bound actin molecule. The target is stretched as a consequence of binding to TRiC and further rearranged in a second step as a consequence of ATP binding; i.e., the mechanism of chaperonins is conserved during evolution.

  • 21. Vincents, Bjarne
    et al.
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Björck, Lars
    Abrahamson, Magnus
    Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding.2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 49, 15540-15549 p.Article in journal (Refereed)
    Abstract [en]

    Streptococcus pyogenes, an important pathogen in humans, secretes an IgG specific endopeptidase named IdeS. To elucidate the mechanism that is responsible for this specificity, we have here characterized the activity of IdeS in detail. Both gamma chains of human IgG or its Fc fragment were cleaved in the hinge region after Gly236 by IdeS, but other proteins or synthetic peptides containing sequences such as the P(4)-P(1) segment in the IgG cleavage site, or long peptides resembling the IgG hinge, were not hydrolyzed at all. This is likely due to a second binding site interacting with the Fc part of IgG. The lack of IdeS activity on peptide substrates necessitated the development of an assay with IgG as the substrate for kinetic studies. IdeS showed a sigmoidal velocity curve at physiological IgG concentrations, and a declining enzyme rate at higher IgG concentrations. This atypical velocity curve suggests product inhibition and/or allosteric control, which again indicates the presence of an exosite involved in substrate binding. The pseudoequilibrium constant for IdeS hydrolysis of IgG was 90 microM. The enzyme exhibited activity in the pH range of 5.1-7.6, with an optimum at pH 6.6. IdeS was stable above pH 10 but not at acidic pH. It exhibited an activity maximum around 37 degrees C and a decreased thermal stability at 42 degrees C. Iodoacetate and iodoacetamide inhibited IdeS, as expected for a cysteine protease, and biochemical evidence verified this classification. E-64 and chicken cystatin, specific inhibitors of family C1 and C13 cysteine proteases, were without effect on enzyme activity, as were class specific serine, aspartic, and metallo protease inhibitors. No significant similarities were found in protein sequence comparisons with known enzyme families, suggesting that IdeS represents a novel family of cysteine proteases.

  • 22. Wolf-Watz, M
    et al.
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Härd, T
    Structure and backbone dynamics of Apo-CBFbeta in solution.2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 38, 11423-11432 p.Article in journal (Refereed)
    Abstract [en]

    Runx proteins constitute a family of mammalian transcription factors that interact with DNA through their evolutionarily conserved Runt domain. CBFbeta, alternatively denoted PEBP2beta, is the non-DNA-binding heterodimer partner and acts to enhance the DNA binding affinity of Runx proteins. Runx proteins and CBFbeta are associated with a variety of biological functions and human diseases; they are, for example, together the most frequent targets for chromosomal rearrangements in acute human leukemias. We have determined the solution structure and characterized the backbone dynamics of C-terminally truncated fragments containing residues 1-141 of CBFbeta. The present apo-CBFbeta structure is very similar to that seen in a Runt-CBFbeta complex. An evaluation of backbone (15)N NMR relaxation parameters shows that CBFbeta is a rigid molecule with high order parameters throughout the backbone; the only regions displaying significant dynamics are a long loop and the C-terminal alpha-helix. A few residues display relaxation behavior indicating conformational exchange on microsecond to millisecond time scales, but only one of these is located at the Runt binding surface. Our structure and dynamics analysis of CBFbeta therefore suggests that the protein binds to Runt without large conformational changes or induced folding ("lock-and-key" interaction). The apo-CBFbeta structure presented here exhibits several significant differences with two other published NMR ensembles of very similar protein fragments. The differences are located in four regions outside of the central beta-barrel, whereas the beta-barrel itself is almost identical in the three NMR structures. The comparison illustrates that independently determined NMR structures may display rather large differences in backbone conformation in regions that appear to be well-defined in each of the calculated NMR ensembles.

  • 23. Yakushevska, A. E.
    et al.
    Keegstra, W.
    Boekema, E. J.
    Dekker, J. P.
    Andersson, Jenny
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Jansson, Stefan
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Ruban, A. V.
    Horton, P.
    The structure of photosystem II in Arabidopsis: localization of the CP26 and CP29 antenna complexes2003In: Biochemistry, ISSN 1520-4995, E-ISSN 0006-2960, Vol. 42, no 3, 608-613 p.Article in journal (Refereed)
    Abstract [en]

    A genetic approach has been adopted to investigate the organization of the light-harvesting proteins in the photosystem II (PSII) complex in plants. PSII membrane fragments were prepared from wild-type Arabidopis thaliana and plants expressing antisense constructs to Lhcb4 and Lhcb5 genes, lacking CP29 and CP26, respectively (Andersson et al. (2001) Plant Cell 13, 1193-1204). Ordered PSII arrays and PSII supercomplexes were isolated from the membranes of plants lacking CP26 but could not be prepared from those lacking CP29. Membranes and supercomplexes lacking CP26 were less stable than those prepared from the wild type. Transmission electron microscopy aided by single-particle image analysis was applied to the ordered arrays and the isolated PSII complexes. The difference between the images obtained from wild type and antisense plants showed the location of CP26 to be near CP43 and one of the light-harvesting complex trimers. Therefore, the location of the CP26 within PSII was directly established for the first time, and the location of the CP29 complex was determined by elimination. Alterations in the packing of the PSII complexes in the thylakoid membrane also resulted from the absence of CP26. The minor light-harvesting complexes each have a unique location and important roles in the stabilization of the oligomeric PSII structure.

  • 24. Zelent, B
    et al.
    Yano, T
    Ohlsson, P-I
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Smith, ML
    Paul, J
    Vanderkooi, JM
    Optical spectra of lactoperoxidase as a function of solvent2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 48, 15953-15959 p.Article in journal (Refereed)
    Abstract [en]

    The iron of lactoperoxidase is predominantly high-spin at ambient temperature. Optical spectra of lactoperoxidase indicate that the iron changes from high-spin to low-spin in the temperature range from room temperature to 20 K. The transformation is independent of whether the enzyme is in glycerol/water or solid sugar glass. Addition of the inhibitor benzohydroxamic acid increases the amount of the low-spin form, and again the transformation is independent of whether the protein is in an aqueous solution or a nearly anhydrous sugar. In contrast to lactoperoxidase, horseradish peroxidase remains high-spin over the temperature excursion in both solvents and with addition of benzohydroxamic acid. We conclude that details of the heme pocket of lactoperoxidase allow ligation changes with temperature that are dependent upon the apoprotein but independent of solvent fluctuations. At low pH, lactoperoxidase shows a solvent-dependent transition; the high-spin form is predominant in anhydrous sugar glass, but in the presence of water, the low-spin form is also present in abundance. The active site of lactoperoxidase is not as tightly constrained at low pH as at neutrality, though the enzyme is active over a wide pH range.

  • 25.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural topology and activation of an initial adenylate kinase-substrate complex2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 6, 1055-1061 p.Article in journal (Refereed)
    Abstract [en]

    Enzymatic activity is ultimately defined by the structure, chemistry and dynamics of the Michaelis complex. There exist a large number of experimentally determined structures between enzymes and substrates or substrate analogues or inhibitors. However, transient, short-lived encounter and equilibrium structures also play fundamental roles during enzymatic reaction cycles. Such structures are inherently difficult to study with conventional experimental techniques. The enzyme adenylate kinase undergoes major conformational rearrangements in response to binding of its substrates ATP and AMP. ATP is sandwiched between two binding surfaces in the closed and active enzyme conformation. Thus, ade-nylate kinase harbors two spatially distant surfaces in the substrate free open conformation of which one is responsible for the initial interaction with ATP. Here, we have performed primarily nuclear magnetic resonance experiments on Escherichia coli adenylate kinase (AKeco) variants that enabled identification of the site responsible for the initial ATP interaction. This allowed a characterization of the structural topology of an initial equilibrium complex between AKeco and ATP. Based on the results it is suggested that the ATP binding mechanism to AKeco is a mixture between "induced fit" and "conformational selection" models. It is shown that ATP is activated in the initial enzyme bound complex since it displays an appreciable rate of non-productive ATP hydrolysis. In summary our results provide novel structural and functional insights into adenylate kinase catalysis.

  • 26.
    Ådén, Jörgen
    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.
    Folding of an unfolded protein by macromolecular crowding in vitro2014In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, no 14, 2271-2277 p.Article in journal (Refereed)
    Abstract [en]

    Protein folding in vivo takes place in a highly crowded environment. The resulting excluded volume forces are thought to stabilize folded forms of proteins. In agreement, many in vitro studies have shown that the presence of macromolecular crowding agents increases the stability of folded proteins but often by only a few kJ per mol. Although it should not matter at what position in the transition between folded and unfolded forms the effect of crowding is employed, there have been no studies assessing whether excluded volume forces alone can correctly fold polypeptides that are mostly unfolded. However, some studies have indicated that the effect of crowding becomes larger the more destabilized the protein is (but still being folded), suggesting that the crowding effect may be exaggerated for unfolded proteins. To address this question directly, we turned to a destabilized mutant of protein L that is mostly unfolded in water but can be folded upon addition of salt. We find that the effect of 200 mg/mL Dextran 20 on the folding equilibrium constant for unfolded protein L (ΔΔGU ≈ 2 kJ mol(-1)) matches the crowding effects found on the folded wild type protein and the mutant when prefolded by salt. This result indicates that the excluded volume effect is independent of starting protein stability and that crowding can shift the reaction toward the folded form when the polypeptide is in the transition region between folded and unfolded states.

  • 27. Öhman, Anders
    et al.
    Lycksell, P O
    Juréus, A
    Langel, U
    Bartfai, T
    Gräslund, A
    NMR study of the conformation and localization of porcine galanin in SDS micelles. Comparison with an inactive analog and a galanin receptor antagonist.1998In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 37, no 25Article in journal (Refereed)
    Abstract [en]

    Galanin is a 29/30-residue neuro-endocrine peptide which performs its many important physiological functions via a membrane-bound receptor. By using two-dimensional proton NMR spectroscopy, complete relaxation matrix analysis, and simulated annealing, the conformation of porcine galanin was determined in a membrane-mimicking solvent containing sodium dodecyl sulfate (SDS) micelles. The final family of calculated structures displays three well-defined beta- or gamma-turn regions, comprising residues 1-5, 7-10, and 24-27, but has otherwise a random conformation. The receptor-interacting N-terminal part, residues 1-5, was found to be best defined with a backbone RMSD value of 0.12 A. The mode of association between galanin and the SDS micelle was determined by observing the broadening effect on proton resonances, when spin-labeled 5- and 12-doxyl stearate molecules were added. It was concluded that galanin is located close to the surface of the micelle with two regions, residues 6-9 and 24-29, as well as two single residues, 18 and 21, reaching out into the aqueous solvent. Additional NMR studies were carried out on an inactive analogue, Ala2-galanin, and an antagonist M40. The results show that the proton resonances of galanin and M40 have identical chemical shifts in the N-terminal receptor-interacting region, indicating similar solution structures in this region. For Ala2-galanin, the same region displays a spectral heterogeneity with chemical shifts clearly different from the other two peptides, indicative of different secondary structures. These results may provide a structural background for the antagonist activity of M40 and the hormonal inactivity of Ala2-galanin, as compared to galanin.

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