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Osmolyte‐induced protein folding free energy changes
Author(s) -
Wu Peng,
Bolen D.W.
Publication year - 2006
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20868
Subject(s) - osmolyte , protein folding , chemistry , folding (dsp implementation) , chaotropic agent , native state , crystallography , protein stability , denaturation (fissile materials) , urea , thermodynamics , biochemistry , physics , electrical engineering , nuclear chemistry , engineering
Upon addition of protecting osmolyte to an aqueous solution of an intrinsically unstructured protein, spectral observables are often seen to change in a sigmoid fashion as a function of increasing osmolyte concentration. Commonly, such data are analyzed using the linear extrapolation model (LEM), a method that defines a scale from 0%–100% folded species at each osmolyte concentration by means of extending pre‐ and post‐folding baselines into the transition region. Defining the 0%–100% folding scale correctly for each osmolyte is an important part of the analysis, leading to evaluation of the fraction of folded protein existing in the absence of osmolytes. In this study, we used reduced and carboxyamidated RNase T1 (RCAM‐T1) as an intrinsically unstructured protein, and determined the thermodynamic stability of RCAM‐T1 induced by naturally occurring osmolytes. Because the folded fraction of the protein population determined by experiments of thermal and urea‐induced denaturation is nonzero in the absence of osmolytes at 15°C, the commonly used LEM can lead to false values of Δ G D→N 0for protein folding due to the arbitrary assumption that the protein is 100% unfolded in the presence of buffer alone. To correct this problem, titration of the protein solution with urea and extrapolating back to zero urea concentration gives the spectral value for 100% denatured protein. With fluorescence as the observable we redefine F / F 0 to F / F 0 extrap= 1.0 and require that the denatured‐state baseline have this value as its intercept. By so doing, the 0%–100% scale‐corrected Δ G D→N 0values of RCAM‐T1 folding in the presence of various osmolytes are then found to be identical, with small error, demonstrating that Δ G D→N 0is independent of the osmolytes used. Such a finding is an important step in validating this quantity derived from the LEM as having the properties expected of an authentic thermodynamic parameter. The rank order of osmolyte efficacies in stabilizing RCAM‐T1 is sarcosine > sucrose > sorbitol > proline > betaine > glycerol. Proteins 2006. © 2006 Wiley‐Liss, Inc.