Abstract
Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This explanation has been questioned and observed effects attributed to weak interactions with other cell components. Here we show conclusively that protein stability is affected by volume exclusion and that the effect is more pronounced when the crowder’s size is closer to that of the protein under study. Accurate evaluation of the volume exclusion effect is made possible by the choice of yeast frataxin, a protein that undergoes cold denaturation above zero degrees, because the unfolded form at low temperature is more expanded than the corresponding one at high temperature. To achieve optimum sensitivity to changes in stability we introduce an empirical parameter derived from the stability curve. The large effect of PEG 20 on cold denaturation can be explained by a change in water activity, according to Privalov’s interpretation of cold denaturation.
Highlights
Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect
This discrepancy might be attributed in part to a basic overestimation of the volume increase upon denaturation[2] and to the relative insensitivity of melting point (Tm) to detect changes in stability
Depending on the combination of thermodynamic parameters, it may happen that the value of thermal unfolding curve (Tm) increases very little or even decreases, whereas thermodynamic stability increases[21,24,36]
Summary
Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This parameter is not very sensitive and, even worse, it can be completely misleading (vide infra) We addressed these compelling questions by undertaking the first detailed study of the influence of crowding on the stability of yeast frataxin (Yfh1), a natural protein that undergoes cold denaturation above water freezing[10]. The nature of the unfolded species changes with temperature, from a strongly hydrated form at lower temperatures to a weakly hydrated one at high temperature[22] Such a situation can be compared with the setup of a sensitive electronic device, say a kind of Wheatstone Bridge[25] for protein stability, because tiny environmental variations will be detected and enhanced as a perturbation of the equilibrium
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
