Differentiation of proteins based on characteristic patterns of association and denaturation in solutions of SDS

Abstract

This paper shows that proteins display an unexpectedly wide range of behaviors in buffers containing moderate (0.1-10 mM) concentrations of SDS (complete unfolding, formation of stable intermediate states, specific association with SDS, and various kinetic phenomena); capillary electrophoresis provides a convenient method of examining these behaviors. Examination of the dynamics of the response of proteins to SDS offers a way to differentiate and characterize proteins. Based on a survey of 18 different proteins, we demonstrate that proteins differ in the concentrations of SDS at which they denature, in the rates of unfolding in SDS, and in the profiles of the denaturation pathways. We also demonstrate that these differences can be exploited in the analysis of mixtures.capillary electrophoresis ͉ surfactant ͉ intermediates ͉ kinetics T his manuscript surveys the range of electrophoretic behaviors observed for proteins in solutions containing SDS at concentrations below those used in SDS͞PAGE. The aggressive conditions used to prepare proteins for characterization by SDS͞PAGE (1) are designed to produce completely denatured, unfolded aggregates of protein and SDS; less forcing conditions have generally been ignored. Because SDS͞PAGE uses forcing conditions, it has failed to reveal the wealth of information available from systems of protein and SDS: information about the kinetics of denaturation; about previously undetected, stable aggregates of protein and SDS with reasonably well defined stoichiometry; and about intermediates along the pathway to the fully denatured aggregates of protein and SDS.Intermediates in the denaturation of some proteins with SDS have been identified: RNase A (2), cytochrome c that had been denatured in acid (3), BSA (4), and mushroom tyrosinase (5). Many proteins have a ''low'' state, in which the protein binds a few molecules of SDS, and ''high'' state, in which the protein binds one molecule of SDS per two amino acids (6, 7). These studies have concentrated mostly on single proteins or on the similarities between proteins and have not demonstrated or exploited the wide variability in behavior of proteins as they are denatured in SDS.We find that proteins show large differences in the concentrations of SDS and in the rates at which they change conformation and unfold in SDS, in the concentrations of SDS at which intermediates form along the unfolding pathway, and in the number of these intermediates. [We use the term ''rate'' to refer to the kinetics of unfolding of the native protein. With this technique, we can only estimate the time scale for unfolding of a protein at a particular concentration of SDS qualitatively, i.e., estimate whether it is shorter, similar, or longer than the time of the capillary electrophoresis (CE) experiment.] These differences among proteins can be exploited to provide the basis for a method of differentiating (and in some instances separating) proteins and information about the relations between their structure and stabilities. We believe that this procedu...

Identifying kinetically stable proteins with capillary electrophoresis

“…Unboiled SVD exhibited two broad faster peaks (7 and 9 min) that suggest the presence of multiple SVD species with different sensitivity to SDS. The existence of more than one type of protein:SDS species was also found by Gudiksen K. L. et al, 9 who observed a mixture of protein:SDS complexes with similar migration times that resulted in broadening of the peak. They suggested that the various peaks result from the different number of SDS molecules binding to various protein conformations.…”

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

“…Interactions of CA with detergents have not been studied extensively, although in recent studies, Whitesides and co-workers 652,657,706,760,761 Early studies of proteins denatured with SDS suggested that two distinct conformations exist in solutions containing low (0.025%) and high (0.1%) (w/v) concentrations of SDS (referred to as the low-and high-binding states, respectively), 762,763 and that both are enzymatically inactive. 757 CD studies indicated that both the high-and low-binding states have conformations that are different from either the native enzyme or the denatured state with GuHCl.…”

Effect of detergent on electromigration of proteins: CE of very low density lipoprotein receptor modules and viral proteins

“…For a number of proteins, the CE patterns showed [10] a sudden shift of the sample peaks at a given SDS concentration (which was not the CMC). Furthermore, the peak shape changed and in some cases multiple peaks were observed.…”