STRUCTURAL ORIGINS OF PRESSURE EFFECTS IN PROTEINS

Cavity;protein folding;pressure;hydration;staphylococcal nuclease;Biophysics

Caro, Jose AHilser, Vincent ;

Johns Hopkins University

关键词: Cavity;    protein folding;    pressure;    hydration;    staphylococcal nuclease;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60519/CARO-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The molecular mechanisms of chemical and heat denaturation of proteins are relatively well established; those of pressure unfolding are not. Volume is the conjugate variable of pressure; it is the fundamental thermodynamic variable that governs the pressure sensitivity of proteins. Cavities that are present in the native state and absent in the unfolded state are thought to contribute significantly to the change in volume upon unfolding (∆V). Staphylococcal nuclease was used to examine the role of cavities systematically. The wild-type protein has a small cavity in its hydrophobic core, comparable in volume to a water molecule. Artificial cavities were generated by substitution of internal hydrophobic residues to Ala. Substitutions of small residues with large ones were used to eliminate the natural cavity. Substitutions to polar residues were used to affect the hydration state of cavities. For 27 variants studied, (a) crystal structures, (b) thermodynamic stabilities using chemical denaturation, and (c) ∆V of unfolding measured by pressure denaturation monitored with Trp-fluorescence and NMR spectroscopy were obtained. In general, the cavities did not affect the structure. The cavities were large enough to hold several waters, but these were only detected in the cavities lined with polar groups. The measured ∆V of variants was always larger than for the wild-type. A near-linear correlation between the ∆V measured experimentally and the one calculated from structures illustrate the importance of cavities in pressure sensitivity. A correlation between measured ∆V and thermodynamic stability (∆G°) suggests that 1 kcal/mol is lost per 11 mL/mol of increased void volume. This study demonstrates that cavities contribute significantly towards the pressure sensitivity of proteins and can modulate the hydration and structural fluctuations of proteins.