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Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Department of Physics, University of Houston, Houston, Texas 77204, United States.
Department of Physics, University of Houston, Houston, Texas 77204, United States.
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2012 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 49, 9836-9845 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2012. Vol. 51, no 49, 9836-9845 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-64042DOI: 10.1021/bi301324yISI: 000312122700009OAI: oai:DiVA.org:umu-64042DiVA: diva2:599791
Available from: 2013-01-22 Created: 2013-01-14 Last updated: 2017-12-06Bibliographically 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.
Keyword
Protein Folding, macromolecular crowding, excluded volume, spectroscopy, stopped-flow
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
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)
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Available from: 2013-11-20 Created: 2013-10-26 Last updated: 2013-11-19Bibliographically approved

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