The structural origin of the decrease in system volume upon protein denaturation by pressure has remained a puzzle for decades. This negative volume change upon unfolding is assumed to arise globally from more intimate interactions between the polypeptide chain and water, including electrostriction of buried charges that become exposed upon unfolding, hydration of the polypeptide backbone and amino acid side chains and elimination of packing defects and internal void volumes upon unfolding of the chain. However, the relative signs and magnitudes of each of these contributing factors have not been experimentally determined. Our laboratory has probed the fundamental basis for the volume change upon unfolding of staphylococcal nuclease (Snase) using variable solution conditions and point mutants of Snase (Royer CA et al., 1993, Biochemistry 32:5222-5232; Frye KJ et al., 1996, Biochemistry 35:10234-10239). Our prior results indicate that for Snase, neither electrostriction nor polar or nonpolar hydration contributes significantly to the value of the volume change of unfolding. In the present work, we investigate the pressure induced unfolding of three point mutants of Snase in which internal cavity size is altered. The experimentally determined volume changes of unfolding for the mutants suggest that loss of internal void volume upon unfolding represents the major contributing factor to the value of the volume change of Snase unfolding. Study holds ProTherm entries: 9526, 9527, 9529 Extra Details: xylose(19 mM) was added in the experiment high pressure; protein folding; staphylococcal nuclease;,volume change
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:38 p.m.
|Number of data points||3|
|Proteins||Thermonuclease ; Thermonuclease|
|Assays/Quantities/Protocols||Experimental Assay: dG pH:5.5, details:Additives ; Experimental Assay: dG details:Additives , pH:7.0 ; Experimental Assay: dG details:Additives xylose(19 mM),, pH:7.0|
|Libraries||Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ|