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. Author manuscript; available in PMC: 2019 Jun 19.
Published in final edited form as: Biophys Chem. 2011 May 31;159(1):129–141. doi: 10.1016/j.bpc.2011.05.020

Fig. 4.

Fig. 4.

A second example of protein allostery in staphylococcal nuclease (SNase) explained by ensemble modulation. A. Experimental acid-unfolding of wild-type SNase monitored by tryptophan fluorescence. A COREX/BEST ensemble-based model of the folding state of the protein describes the observed data better than a static structure model using Poisson–Boltzmann electrostatics, as previously described. [54] The gray-shaded region indicates conditions where the protein’s conformational fluctuations are predominantly local. B. Experimental vs. computed proton binding in SNase. Again, the COREX/BEST ensemble-based model reproduces the observed data more faithfully than does a model based on only the fully folded and fully unfolded states of the protein, as previously described [54]. C. Global coupling parameters, GCP, were used to investigate the microscopic origins of the pH effects described in panels A and B. High GCP values (yellow and red on the accompanying scale) indicate residues whose pH titration is dependent on conformational redistribution of the ensemble. Only a subset of all ionizable residues exhibit high GCP values and are predicted to govern the energetics of the acid unfolding reaction. As previously described [54], the GCP at each residue j in the protein was defined as a function of pH: GCP(pH)j=(Z(pH)f,j−<Z(pH)j>)×(Z(pH)nf,j−<Z(pH)j>). In this equation, the values Z(pH)f,j and Z(pH)nf,j are the pH dependent protonation states of residue j in the fully folded and fully unfolded states, respectively. <Z(pH)j> is the ensemble-weighted protonation value. D. The effects of point mutations on the stability of SNase at pH 7; ΔΔGpH7 indicates the difference in stability between the wild-type protein and the labeled mutants. The black curve describes the expected dependence; for mutants that fall on the curve (blue points), the substituted amino acid is not coupled to the global unfolding transition. Mutants that do not fall on the curve (red labeled points) are often in the subset identified in panel C, suggesting that the pH-dependent energetics of SNase folding are controlled by ensemble and not static structure effects. This figure and accompanying results are adapted from Whitten, et al.[54]. Copyright (2005) National Academy of Sciences, USA.