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Electronic Energy Migration/Transfer as a Tool to Explore Biomacromolecular Structures
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Lennart B.-Å. Johansson)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions.

The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso.

The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding.

We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur.

In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence. 

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2014. , 55 p.
Keyword [en]
Fluorescence, electronic energy transfer, two-photon excitation, small ribosomal protein S16, macromolecular crowding, dextran 20
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-86794ISBN: 978-91-7601-021-1 (print)OAI: oai:DiVA.org:umu-86794DiVA: diva2:704092
Public defence
2014-04-04, KBC-huset, KB3B1, Umeå universitet, Linnaeus Väg, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 550021104
Available from: 2014-03-12 Created: 2014-03-11 Last updated: 2014-03-11Bibliographically approved
List of papers
1. On the analyses of fluorescence depolarisation data in the presence of electronic energy migration.: II. Applying & Evaluating Two-Photon Excited Fluorescence
Open this publication in new window or tab >>On the analyses of fluorescence depolarisation data in the presence of electronic energy migration.: II. Applying & Evaluating Two-Photon Excited Fluorescence
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2012 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 14, 1917-1922 p.Article in journal (Refereed) Published
Abstract [en]

Electronic energy migration within a bifluorophoric molecule has been studied by time-resolved two-photon excited (TPE) fluorescence depolarisation experiments. Data were analysed by using a recently developed quantitative approach [Opanasyuk, O. & Johansson, L. B.-Å., On the Analyses of Fluorescence Depolarisation Data in the Presence of Electronic Energy Migration. I. Theory & General Description. Phys. Chem. Chem. Phys., Submitted.]. The energy migration occurs between the 9-anthrylmethyl groups of the bifluorophoric molecule, bis-(9-anthrylmethylphosphonate) bisteroid. These groups undergo local reorientations, while overall tumbling of the bisteroid is strongly hampered in the used viscous solvent, 1,2-propanediol. To solely obtain information about local reorientations of the 9-anthrylmethyl group, also the mono-(9-anthrylmethylphosphonate) bisteroid was studied, which enabled modelling of the ordering potential shape. The analysis of data is partly performed in the Fourier domain and the best-fit parameters are determined by using an approach based on a Genetic Algorithm. The energy migration process was described by an extended Förster theory (EFT). A reasonable value of the distance between the 9-anthrylmethyl groups is found, as well as for the mutual orientation of the ordering potentials. Furthermore, values of the two-photon tensor components were obtained.

Keyword
Extended Förster theory, Genetic algorithms, Monte Carlo simulations, two-photon excitation
National Category
Physical Chemistry Atom and Molecular Physics and Optics
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-50049 (URN)10.1039/C2CP23177H (DOI)
Available from: 2011-11-24 Created: 2011-11-24 Last updated: 2017-12-08Bibliographically approved
2. Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state
Open this publication in new window or tab >>Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state
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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 379, no 4, 845-858 p.Article in journal (Refereed) Published
Abstract [en]

Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16meso) and hyperthermophilic (S16thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔCp0) of 6.4 and 3.3 kJ mol− 1 K− 1, respectively. A strong linear correlation between ΔCp0 and melting temperature (Tm) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16meso folds through an apparent two-state model, whereas S16thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16thermo is more compact relative to S16meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16thermo that appears to account for the large difference in quantified ΔCp0 values and, in turn, parts of the observed extreme thermal stability of S16thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.

Keyword
ribosomal protein S16, residual structure, protein folding, thermostability, ΔCp0
Identifiers
urn:nbn:se:umu:diva-9439 (URN)10.1016/j.jmb.2008.04.007 (DOI)
Available from: 2008-05-29 Created: 2008-05-29 Last updated: 2017-12-14Bibliographically approved
3. Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding
Open this publication in new window or tab >>Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding
2013 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 3, 694-704 p.Article in journal (Refereed) Published
Abstract [en]

Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.

Place, publisher, year, edition, pages
Cell Press, 2013
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-65815 (URN)10.1016/j.bpj.2012.12.020 (DOI)
Available from: 2013-02-12 Created: 2013-02-12 Last updated: 2017-12-06Bibliographically approved
4. Effects of macromolecular crowding on folded protein homologs: global versus local consequences
Open this publication in new window or tab >>Effects of macromolecular crowding on folded protein homologs: global versus local consequences
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Proteins function in cellular environments that are crowded with other biomolecules. This reduction of available space may change biophysical properties of proteins as compared to dilute solutions in vitro. Here we have investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16Thermo) and mesophilic (S16Meso) homologs of the ribosomal protein S16. First, as expected for an excluded volume effect, the thermal stability increased for S16Meso in the presence of dextran 20. In accord with a dominating entropic effect, chemical denaturation experiments at different fixed temperatures showed that the crowding effect was temperature independent. Far-UV circular dichroism spectra did not reveal any secondary structural changes in the folded proteins in presence of dextran 20. However, Förster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY in S16Meso depend on the level of crowding agent. The BODIPY-group was attached at three specific positions in S16Meso. All S16Meso variants exhibited a decrease in the average distance up to 100 mg/mL dextran 20, indicating folded-state compaction, whereas the change in distance became anisotropic at higher dextran concentrations. In contrast, the two S16Thermo mutants, that are thermodynamically more stable than the mesophilic variants, did not show any change in distance upon increasing dextran 20 concentrations. Notably, the BODIPY fluorescence quantum yields and lifetimes of the two homologs decreased gradually in the presence of dextran 20. To investigate the origin of this, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labelled dextran. The results reveal quenching between tyrosine and BODIPY at all concentrations of Tyr-dextran. The data also suggests that dynamic local interactions between protein and crowding agent can occur at some conditions.

Keyword
Excluded volume, Fluorescence, Tryptophan-BODIPY, Förster resonance energy transfer, dextran 20, small ribosomal protein S16
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-86791 (URN)
Funder
Swedish Research Council, 550021104
Available from: 2014-03-11 Created: 2014-03-11 Last updated: 2014-03-11

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