A large-conductance mechanosensitive channel in E. coli encoded by mscL alone

Sukharev, Sergei; Blount, Paul; Martinac, Boris; Blattner, Frederick R.; Kung, Ching

doi:10.1038/368265a0

Cited by 677 publications

(593 citation statements)

References 11 publications

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“…1. A challenge for the chemical synthesis of Ec-MscL and Tb-MscL was the absence of cysteine residues in the WT sequence (21,22); introduction of cysteine residues at ligation sites results in the presence of nonnative sulfhydryl groups in the final protein. For the synthesis of Ec-MscL, glutamine 56 (Q56) and asparagine 103 (N103) were changed to cysteine, based on unpublished data (J.A.M.)…”

Section: Resultsmentioning

confidence: 99%

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

Clayton

Shapovalov

Maurer

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

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

Clayton

Shapovalov

Maurer

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Fluid flow can impart a shear stress that will augment mass transport [135][136][137]. Cellular internalization of lipoplexes was increased more than nine-fold at 2.3 dyn/cm 2 compared to the static condition, but decreased again for higher shear stresses due to disruption of lipoplex-cell binding [138].…”

Section: Microenvironment and Gene Transfermentioning

confidence: 97%

Matrices and scaffolds for DNA delivery in tissue engineering

Laporte¹,

Shea²

2007

Advanced Drug Delivery Reviews

240

208

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Regenerative medicine aims to create functional tissue replacements, typically through creating a controlled environment that promotes and directs the differentiation of stem or progenitor cells, either endogenous or transplanted. Scaffolds serve a central role in many strategies by providing the means to control the local environment. Gene delivery from the scaffold represents a versatile approach to manipulating the local environment for directing cell function. Research at the interface of biomaterials, gene therapy, and drug delivery has identified several design parameters for the vector and the biomaterial scaffold that must be satisfied. Progress has been made towards achieving gene delivery within a tissue engineering scaffold, though the design principles for the materials and vectors that produce efficient delivery require further development. Nevertheless, these advances in obtaining transgene expression with the scaffold have created opportunities to develop greater control of either delivery or expression and to identify the best practices for promoting tissue formation. Strategies to achieve controlled localized expression within the tissue engineering scaffold will have broad application to the regeneration of many tissues, with great promise for clinical therapies.

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“…A change in the perception of lipids as actively regulating protein function came from the study of a pair of bacterial mechanosensitive channels, the heptameric mechanosensing channel of small conductance (MscS) [2,3] and the pentameric mechanosensing channel of large conductance (MscL) [4,5]. These proteins are controlled by changes in the lipid bilayer that arise from increased pressure inside the cell (known as turgor).…”

Section: Introductionmentioning

confidence: 99%

Spectator no more, the role of the membrane in regulating ion channel function

Pliotas

Naismith

2017

Current Opinion in Structural Biology

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A pressure gradient across a curved lipid bilayer leads to a lateral force within the bilayer. Following ground breaking work on eukaryotic ion channels, it is now known that many proteins sense this change in the lateral tension and alter their functions in response. It has been proposed that responding to pressure differentials may be one of the oldest signaling mechanisms in biology. The most well characterized mechanosensing ion channels are the bacterial ones which open when the pressure differential hits a threshold. Recent studies on one of these channels, MscS, have developed a simple molecular model for how they sense and adapt to pressure.Biochemical and structural studies on mechanosensitive channels from eukaryotes have disclosed pressure sensing mechanisms. In this review, we highlight these findings and discuss the potential for a general model for pressure sensing.3

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A large-conductance mechanosensitive channel in E. coli encoded by mscL alone

Cited by 677 publications

References 11 publications

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

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

Matrices and scaffolds for DNA delivery in tissue engineering

Spectator no more, the role of the membrane in regulating ion channel function

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