Interaction of a membrane protein with its surrounding lipid bilayer : studies with the mechanosensitive channel MscL

Abstract

mechanosensitive (MS) channels open in response to membrane stretch.  The most studied of the MS channels is the bacterial mechanosensitive ion channel of large conductance, MscL.  Interactions between the lipid bilayer and MscL are particularly important since MscL opens on increasing tension in model systems containing just lipid and MscL, so that membrane tension must be transduced directly from the lipid molecules to the protein.  Key to understanding how stretching of the lipid bilayer leads to opening of MscL is understanding how MscL interacts with the lipid bilayer.  Site directed mutagenesis and fluorescence spectroscopy were used to define how the MscL channel interacts with the lipid bilayer that surrounds it in the membrane.  Single Trp-containing mutants of Mycobacterium tuberculosis MscL were reconstituted into lipid bilayers of defined composition.  Fluorescence emission maxima were used to identify the transmembrane region of MscL in bilayers of dioleoylphosphatidylcholine (di(C18:1)PC) and the levels of fluorescence quenching by brominated lipid gave lipid binding constants for MscL in the vicinity of the Trp groups.  Introducing a lysine residue into the central pore of MscL generated a gain of function phenotype, thereby allowing the lipid-protein interactions of both the open and closed channel to be determined.  Preferential binding was observed with phosphatidylcholines possessing fatty acyl chains with a length of C16 for the closed channel and C14 for the open channel, suggesting the open channel has thinned by 4 Å.  All lipid head groups bind equally well to MscL on the periplasmic side of the membrane.  In contrast, anionic lipids bind with significantly greater affinity on the cytoplasmic side of MscL, with two classes of binding site involving the conserved charge cluster Arg-98, Lys-99 and Lys-100.  This binding site is broken up when the channel forms an open structure.

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