title: The fluorescence detected guanidine hydrochloride equilibrium denaturation of wild-type staphylococcal nuclease does not fit a three-state unfolding model.
abstract: A three-state equilibrium unfolding of a protein can be difficult to detect if two of the states fail to differ in some easily measurable way. It has been unclear whether staphylococcal nuclease unfolds in a two-state fashion, with only the native and denatured states significantly populated at equilibrium, or in a three-state manner, with a well-populated intermediate. Since equilibrium unfolding experiments are commonly used to determine protein stability and the course of denaturation are followed by changes in the fluorescence which has difficulty in distinguishing various states, this is a potential problem for many proteins. Over the course of twenty years we have performed more than one hundred guanidine hydrochloride equilibrium denaturations of wild-type staphylococcal nuclease; to our knowledge, a number of denaturations unrivaled in any other protein system. A careful examination of the data from these experiments shows no sign of the behavior predicted by a three-state unfolding model. Specifically, a three-state unfolding should introduce a slight, but characteristic, non-linearity to the plot of stability versus denaturant concentration. The average residuals from this large number of repeated experiments do not show the predicted behavior, casting considerable doubt on the likelihood of a three-state unfolding for the wild-type protein. The methods used for analysis here could be applied to other protein systems to distinguish a two-state from a three-state denaturation.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, CHEM 119, Fayetteville, AR 72701, USA.
authors.forename: Deepika
authors.initials: D
authors.lastname: Talla
authors.forename: Wesley E
authors.initials: WE
authors.lastname: Stites
mesh_terms: D005453:Fluorescence; D019791:Guanidine / Q000737:chemistry*; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability
chemical_list: D008836:Micrococcal Nuclease; D019791:Guanidine