The Effect of Detergent, Temperature, and Lipid on the Oligomeric State of MscL Constructs: Insights from Mass Spectrometry
The mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology.
© 2015 Elsevier Ltd. Received: March 11, 2015; Revised: April 23, 2015; Accepted: April 27, 2015; Published: May 21, 2015. This work was supported by an ERC Advanced Investigator Award (26851) (IMPRESS), a Royal Society Research Professorship, a Medical Research Council Program Grant (98101) and an NIH grant GM084211. Author Contributions: E.R., T.A.W., I.L., and A.L. performed the experiments and analyzed the data. E.R., T.A.W., A.L., D.C.R, and C.V.R designed the study. M.T.M. developed the deconvolution software and provided analysis support for this study. E.R. and C.V.R. wrote the article with contributions from all other authors. Supplemental Information includes three tables and ten figures and can be found with this article online at http://dx.doi.org/10.1016/j.chembiol.2015.04.016.
Supplemental Material - mmc1.pdf
Accepted Version - nihms-1028275.pdf