Protein stability: functional dependence of denaturational Gibbs energy on urea concentration.
Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.
Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India.
R
R
Gupta
F
F
Ahmad
D000818:Animals; D002417:Cattle; D002645:Chickens; D003574:Cytochrome c Group / Q000737:chemistry; D004529:Egg White / Q000032:analysis; D019791:Guanidine / Q000737:chemistry; D006736:Horses; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry*
D003574:Cytochrome c Group; D011506:Proteins; D014508:Urea; D012259:Ribonuclease, Pancreatic; D009113:Muramidase; D019791:Guanidine