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Macromolecular crowding tunes folding landscape of parallel α/β protein, apoflavodoxin
University of Texas MD Anderson, Houston and Rice University, Houston, Texas United States .
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Chemical Biological Center)
Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, United States. (Biological Chemistry)
2011 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, no 4, 646-648 p.Article in journal (Refereed) Published
Abstract [en]

Proteins normally fold in crowded cellular environments. Here we use a set of Desulfovibrio desulfuricans apoflavodoxin variants to assess-with residue-specific resolution-how apoflavodoxin's folding landscape is tuned by macromolecular crowding. We find that, under crowded conditions, initial topological frustration is reduced, subsequent folding requires less ordering in the transition state, and β-strand 1 becomes more important in guiding the process. We propose that conditions more closely mimicking the cellular environment make the ensemble of unfolded conformations less expanded, resulting in a folding funnel that is smoother and narrower.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2011. Vol. 133, no 4, 646-648 p.
National Category
Chemical Sciences
URN: urn:nbn:se:umu:diva-38759DOI: 10.1021/ja107638ePubMedID: 21175168OAI: diva2:381554
Available from: 2010-12-28 Created: 2010-12-28 Last updated: 2013-11-19Bibliographically approved
In thesis
1. Effects of Macromolecular Crowding on Protein Folding: - in-vitro equilibrium and kinetic studies on selected model systems
Open this publication in new window or tab >>Effects of Macromolecular Crowding on Protein Folding: - in-vitro equilibrium and kinetic studies on selected model systems
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein folding is the process during which an extended and unstructured polypeptide converts to its compact folded structure that is most often the functional state. The process has been characterized extensively in dilute buffer in-vitro during the last decades but the actual biological place for this process is the inside of living cells. The cytoplasm of a cell is filled with a plethora of different macromolecules that together occupy up to 40% of the total volume. This large amount of macromolecules restricts the available space to each individual molecule, which has been termed macromolecular crowding. Macromolecular crowding results in excluded volume effects and also increases chances for non-specific interactions. Macromolecular crowding should favor reactions that lead to a decrease in the total occupied volume by all molecules, such as folding reactions. Theoretical models have predicted that the stability of protein folded states should increase in presence of macromolecular crowding due to unfavorable effects on the extended unfolded state. To understand protein folding and function in living systems, we need to have a defined quantitative link between in-vitro dilute conditions (where most biophysical experiments are made) and in-vivo crowded conditions. An important question is thus how macromolecular crowding modifies the biophysical properties of a protein.

The work underlying this thesis focused on how macromolecular crowding tunes protein equilibrium stability and kinetic folding processes. To mimic the crowded cellular environment, synthetic sugar-based polymers (Dextrans of different sizes and Ficoll 70) were used as crowding agents (crowders) in controlled in-vitro experiments. In contrast to previous studies which often have focused on one protein and one crowder at a time, the goal here was to make systematic analyses of how size, shape and concentration of the crowders affect both equilibrium and kinetic properties of structurally-different proteins. Three model proteins (cytochrome c, apoazurin and apoflavodoxin) were investigated under crowding by Ficoll 70 and different-size Dextrans, using various spectroscopic techniques such as far-UV circular dichroism and intrinsic tryptophan fluorescence. Thermodynamic models were applied to explain the experimental results.

It was discovered that equilibrium stability of all three proteins increased in presence of crowding agents in a crowder concentration dependent manner. The stabilization effect was around 2-3 kJ/mol, larger for the various Dextrans than for Ficoll 70 at the same g/l, but independent of Dextran size (in the range 20 to 70 kDa). To further investigate the cause for the stabilization a theoretical crowding model was applied. In this model, Dextran and Ficoll were modeled as elongated rods and the protein was represented as a sphere, where the folded sphere representation was smaller than the unfolded sphere representation. It is notable that the observed stability changes could be reproduced by this model taking only steric interactions into account. This correlation showed that when using sugar-based crowding agents, excluded volume effects could be studied in isolation and there were no contributions from nonspecific interactions.

Time-resolved experiments with apoazurin and apoflavodoxin revealed an increase in the folding rate constants while the unfolding rates were invariant in the presence of crowding agents. For apoflavodoxin and cytochrome c, the presence of crowding agents also altered the folding pathway such that it became more homogeneous (cytochrome c) and it gave less misfolding (apoflavodoxin). These results showed that macromolecular crowding restricts the conformational space of the unfolded polypeptide chain, makes the conformations more compact which, in turn, eliminates access to certain pathways.

The results from kinetic and equilibrium measurements on three model proteins, together with available data from the literature, demonstrate that macromolecular crowding effects due to volume exclusion are in the order of a few kJ/mol. Considering the numerous concentration balances and cross-dependent reactions of the cellular machinery, small changes in energetics/kinetics of the magnitudes found here can still have dramatic consequences for cellular fitness. In fact local and transient changes in macromolecular crowding levels may be a way to tune biochemical reactions without invoking gene expression. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2013. 85 p.
Protein Folding, macromolecular crowding, excluded volume, spectroscopy, stopped-flow
National Category
Chemical Sciences
Research subject
urn:nbn:se:umu:diva-82059 (URN)978-91-7459-764-6 (ISBN)
Public defence
2013-12-11, KBC-Huset, KB3B1, Umeå universitet, Umeå, 10:00 (English)
Available from: 2013-11-20 Created: 2013-10-26 Last updated: 2013-11-19Bibliographically approved

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