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

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  • Daniel E Otzen
  • Jannik Nedergaard Pedersen, Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO)
  • ,
  • Arun Kumar Somavarapu
  • Anders Clement, Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO)
  • ,
  • Ming Ji, Johns Hopkins University Bayview Proteomic Center, Department of Medicine, Johns Hopkins University School of Medicine
  • ,
  • Emil Hartvig Petersen, Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO)
  • ,
  • Jan Skov Pedersen
  • Nicholas P Schafer, Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO)

Empirically α-helical membrane protein folding stability in surfactant micelles can be tuned by varying the mole fraction MFSDS 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 behaviour. Here, we use CL to investigate membrane protein folding in mixed DDM-SDS micelles. Labelling kinetics of the intramembrane protease GlpG are consistent with simple two-state unfolding-and-exchange rates for 7 single-Cys GlpG variants over most of the explored MFSDS range, along with exchange from the native state at low MFSDS (which inconveniently precludes measurement of unfolding kinetics under native conditions). However, for two mutants, labelling rates decline with MFSDS at 0-0.2 MFSDS (i.e. native conditions). Thus an increase in MFSDS 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 Cys labelling (based on computational simulations) along with changes in size and shape of the mixed micelle with changing MFSDS (based on SAXS studies). These non-linear impacts on protein stability highlights a multi-faceted 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
ISSN0006-3495
DOIs
Publication statusE-pub ahead of print - 6 Aug 2021

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