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Effects of macromolecular crowding on folded protein homologs: global versus local consequences
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Lennart Johansson)
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Pernilla Wittung-Stafshede)
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
(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 [en]
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: urn:nbn:se:umu:diva-86791OAI: oai:DiVA.org:umu-86791DiVA: diva2:704065
Funder
Swedish Research Council, 550021104
Available from: 2014-03-11 Created: 2014-03-11 Last updated: 2014-03-11
In thesis
1. Electronic Energy Migration/Transfer as a Tool to Explore Biomacromolecular Structures
Open this publication in new window or tab >>Electronic Energy Migration/Transfer as a Tool to Explore Biomacromolecular Structures
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
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:nbn:se:umu:diva-86794 (URN)978-91-7601-021-1 (ISBN)
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

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