Structural thermodynamics of a random coil protein in guanidine hydrochloride.
An important problem in protein folding is to understand the relationship between the structure of a denatured ensemble and its thermodynamics. Using 0 - 6M GdnHCl at fixed pH, we evaluated dimensional changes of an extensively denatured ensemble along with a thermodynamic parameter (Deltaupsilon) that monitors the proton inventory of the ensemble. Reduced and carboxyamidated ribonuclease A (RCAM) is a member of a class of disulfide-free RNase A molecules believed to be random coils (extensively denatured) in aqueous solution. Because GdnHCl interacts more favorably with the protein than water does, this denaturant is observed to increase the Stokes radius of the random coil, with the greatest Stokes radius change occurring in the 0 - 1.5M GdnHCl range. Measurement of the degree of protonation (proton inventory) of the ensemble as a function of GdnHCl at the fixed pH shows that the thermodynamic character of the ensemble also changes markedly in the 0 - 1.5M GdnHCl range, but with little or no change beyond 1.5M GdnHCl. To obtain denaturant-independent DeltaG degrees (N-D) values, the linear extrapolation method (LEM) requires the thermodynamic character of the native and denatured ensembles to be invariant in the transition zone. The results reported here indicate that proteins with a transition midpoint in the 0 - 1.5M GdnHCl range will not give denaturant-concentration independent DeltaG degrees (N-D) values. Such LEM-derived DeltaG degrees (N-D) quantities are a property of the protein and the denaturant, a condition that considerably limits their value in understanding structural energetics.
Yang
M
M
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 7755-1052, USA.
Ferreon
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Bolen
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D019791:Guanidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013816:Thermodynamics
D012259:Ribonuclease, Pancreatic; D019791:Guanidine