Residue-Level Interrogation of Macromolecular Crowding Effects on Protein Stability

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 06/2008; 130(21):6826-30. DOI: 10.1021/ja8005995
Source: PubMed


Theory predicts that macromolecular crowding affects protein behavior, but experimental confirmation is scant. Herein, we report the first residue-level interrogation of the effects of macromolecular crowding on protein stability. We observe up to a 100-fold increase in the stability, as measured by the equilibrium constant for folding, for the globular protein chymotrypsin inhibitor 2 (CI2) in concentrations of the cosolute poly(vinylpyrrolidone) (PVP) that mimic the protein concentration in cells. We show that the increased stability is caused by the polymeric nature of PVP and that the degree of stabilization depends on both the location of the individual residue in the protein structure and the PVP concentration. Our data reinforce the assertion that macromolecular crowding stabilizes the protein by destabilizing its unfolded states.

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Available from: Conggang Li, Oct 07, 2015
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    • "In vitro biochemical and in silico approaches also lack the cellular milieu that is essential for protein dynamics. Post-translational modifications and intracellular crowding of macromolecules, including chaperones, affect protein interactions [21] that, in turn, influence the folding rates [22], stability, and function of proteins [23] [24] [25]. Ideally, physiologically relevant measurements of proteostasis should be carried out in live cells. "
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    ABSTRACT: Evidence of protein misfolding and proteostasis decline is a common feature of many neurodegenerative diseases. However, modeling the complexity of proteostasis and the global cellular consequences of its disruption is a challenge, particularly in live neurons. Although conventional approaches, based on population measures and single "snapshots", can identify cellular changes during neurodegeneration, they fail to determine if these cellular events drive cell death or act as adaptive responses. Alternatively, a "systems" cell biology approach known as longitudinal survival analysis enables single neurons to be followed over the course of neurodegeneration. By capturing the dynamics of misfolded proteins and the multiple cellular events that occur along the way, the relationship of these events to each other and their importance and role during cell death can be determined. Quantitative models of proteostasis dysfunction may yield unique insight and novel therapeutic strategies for neurodegenerative disease.
    FEBS letters 03/2013; 587(8). DOI:10.1016/j.febslet.2013.02.043 · 3.17 Impact Factor
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    • "Extending these studies, Pielak and co-workers [7] [8] [9] investigated the effects of both a polymer crowder, poly(vinylpyrrolidone) (PVP), and two protein crowders, lysozyme and bovine serum albumin (BSA), on the folding stability of a small protein chymotrypsin inhibitor 2 (CI2), a known reversible two-state folder [10]. In line with the other studies, the polymer crowder PVP was found to have a moderate stabilizing effect on CI2, but the two protein crowders were found to be destabilizing, leading to the suggestion that polymers and proteins behave differently as crowding agents. "
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    ABSTRACT: Recently a polymer crowder and two protein crowders were found to have opposite effects on the folding stability of chymotrypsin inhibitor 2 (CI2), suggesting that they interact differently with CI2. Here we propose that all the macromolecular crowders act similarly, with an entropic component favoring the folded state and an enthalpic component favoring the unfolded state. The net effect is destabilizing below a crossover temperature but stabilizing above it. This general trend is indeed observed in recent experiments and hints experimental temperature as a reason for the opposite crowding effects of the polymer and protein crowders.
    FEBS letters 01/2013; 587(5). DOI:10.1016/j.febslet.2013.01.030 · 3.17 Impact Factor
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    • "The observed increase in k cat with addition of macromolecular crowding agent implies that the excluded volume effect makes connectivity in the protein medium stronger and/or reduces copper-site reorganization energy (by affecting solvent dynamics) upon redox change. In accord, internal protein dynamics have been shown to change with crowding [25] [30] and, for Fet3p, this Fig. 2 "
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    ABSTRACT: Enzymes catalyze biochemical reactions in highly crowded environments where the amount of macromolecules may occupy up to 40% of the volume. Here we report how cell-like conditions tune catalytic parameters for the monomeric multi-copper oxidase, Saccharomyces cerevisiae Fet3p, in vitro. At low amounts of crowding agent, we detect increases in both of K(M) (weaker substrate binding) and k(cat) (improved catalytic efficiency), whereas at higher crowding levels, both parameters were reduced. Presence of crowding agents does not affect Fet3p structural content but increases thermal resistance. The observations are compatible with ordering of a non-optimal substrate-binding site and restricted internal dynamics as a result of excluded volume effects making the protein less structurally 'strained'.
    Biochimica et Biophysica Acta 11/2009; 1804(4):740-4. DOI:10.1016/j.bbapap.2009.11.013 · 4.66 Impact Factor
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