Cys-labelling kinetics of membrane protein GlpG: a role for specific SDS binding and micelle changes?

Daniel E Otzen, Jannik Nedergaard Pedersen, Arun Kumar Somavarapu, Anders Clement, Ming Ji, Emil Hartvig Petersen, Jan Skov Pedersen, Sinisa Urban, Nicholas P Schafer

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Empirically, α-helical membrane protein folding stability in surfactant micelles can be tuned by varying the mole fraction MF SDS of anionic (sodium dodecyl sulfate (SDS)) relative to nonionic (e.g., dodecyl maltoside (DDM)) surfactant, but we lack a satisfying physical explanation of this phenomenon. Cysteine labeling (CL) has thus far only been used to study the topology of membrane proteins, not their stability or folding behavior. Here, we use CL to investigate membrane protein folding in mixed DDM-SDS micelles. Labeling kinetics of the intramembrane protease GlpG are consistent with simple two-state unfolding-and-exchange rates for seven single-Cys GlpG variants over most of the explored MF SDS range, along with exchange from the native state at low MF SDS (which inconveniently precludes measurement of unfolding kinetics under native conditions). However, for two mutants, labeling rates decline with MF SDS at 0–0.2 MF SDS (i.e., native conditions). Thus, an increase in MF SDS seems to be a protective factor for these two positions, but not for the five others. We propose different scenarios to explain this and find the most plausible ones to involve preferential binding of SDS monomers to the site of CL (based on computational simulations) along with changes in size and shape of the mixed micelle with changing MF SDS (based on SAXS studies). These nonlinear impacts on protein stability highlights a multifaceted role for SDS in membrane protein denaturation, involving both direct interactions of monomeric SDS and changes in micelle size and shape along with the general effects on protein stability of changes in micelle composition.

Original languageEnglish
JournalBiophysical Journal
Pages (from-to)4115-4128
Publication statusPublished - Sept 2021


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