Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

Abstract

Total chemical protein synthesis was used to generate multimilligram quantities of the mechanosensitive channel of large conductance from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (Tb-MscL). Cysteine residues introduced to allow chemical ligation were masked with cysteine-reactive molecules, resulting in side chain functional groups similar to those of the wild-type protein. Synthetic channel proteins were transferred to 2,2,2-trifluoroethanol and reconstituted into vesicle membranes. Fluorescent imaging of vesicles showed that channel proteins were membrane-localized. Single-channel recordings showed that reconstituted synthetic Ec-MscL has conductance, pressure dependence, and substate distribution similar to those of the recombinant channel. Reconstituted synthetic Tb-MscL also displayed conductance and pressure dependence similar to that of the recombinant protein. Possibilities for the incorporation of unnatural amino acids and biophysical probes, and applications of such synthetic ion channel analogs, are discussed. R ecently, ion channel research has been aided by the discovery of bacterial ion channels that share many key features of mammalian channels (1-3). Some of these bacterial channel proteins are smaller and more amenable to expression and purification than mammalian channels. Importantly, bacterial channels can often be reconstituted into vesicles and other synthetic membrane systems, allowing detailed functional characterization (4-6). Furthermore, several bacterial channels have been described at atomic resolution by x-ray crystallography (7-11), providing opportunities for correlating structure and function in a key class of membrane protein.Although the preparation of many medium-sized watersoluble proteins by chemical ligation is now a routine procedure (12)(13)(14), the synthesis of ion channels and other membrane proteins presents unique challenges. Many hydrophobic peptides have sequences that couple inefficiently, meaning that the synthesis of each segment must be optimized. Hydrophobic peptides also have limited solubility and tend to aggregate. Furthermore, after synthesis is complete the native oligomeric protein must be formed from unfolded monomers. However, recent advances in chemical ligation methodologies have permitted the semisynthesis (15) and the total chemical synthesis of membrane proteins, including the 97-residue type III (single membrane-spanning segment) M2 protein of the influenza virus (16). The synthetic M2 protein assembled into the native tetrameric form on reconstitution into dodecylphosphocholine micelles.Here, we describe the chemical synthesis of two polytopic membrane proteins, the mechanosensitive ion channels from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (TbMscL), and the vesicle reconstitution of these channels into a form functionally similar to that of the recombinant protein.Multimilligram quantities of Ec-MscL and Tb-MscL were generated by optimizing synthesis, purification, and ligation protocols previously developed fo...

Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit

“…Chemical protein synthesis has previously been used to incorporate D-amino acids (Bang et al 2006a), backbonemodified residues (Lu et al 1999;Johnson and Kent 2006b), and spectroscopic probes (Cowburn et al 2004) in a number of systems, including the MscL (Clayton et al 2004) and KcsA (Valiyaveetil et al 2004) ion channels. The major challenge in most of these studies is in the synthesis of the modified protein itself.…”

Mechanosensitive channel of large conductance

“…In addition, bacterial growth assays are only suitable for MscL mutants produced in vivo. However, MscL has been synthesized chemically via solid-phase synthesis and native chemical ligation [22]. A highthroughput, cell-free assay would be superior to current techniques because it would allow for more rapid screening of mutants as well as characterization of mutants synthesized in vitro.…”

Mechanical properties of lipid bilayers and regulation of mechanosensitive function

“…110,144,145 As a final point, the ultimate goal will be the design of "prebiotic peptides" with a desirable osmo-valve activity in a precell context. Considering that oligomerization was found to be inherent to the primary sequence of MscL for its in vitro synthesis, 18 and since chemically synthesized MscL proteins can be directly reconstituted in liposomes with high efficiency, 19,20 it becomes tempting to study alternative "prebiotic" sequences 146 in the context of model protocells. Indeed, the role of ancient peptides responding to membrane tension has been claimed as a determining factor allowing protocells to gain volume-control and osmoregulation.…”

“…17 Overall, these observations suggest that MS channels have evolved responding to a common stimulus at the level of membrane mechanical properties. In this context, the remarkable in vitro chemical synthesis and oligomerization of bacterial MscL proteins into functional channels [18][19][20] opens attractive new research perspectives. In addition, the use of pore-forming peptides has provided important information for this approach.…”