Conversion of a mechanosensitive channel protein from a membrane-embedded to a water-soluble form by covalent modification with amphiphiles.
Covalent modification of integral membrane proteins with amphiphiles may provide a general approach to the conversion of membrane proteins into water-soluble forms for biophysical and high-resolution structural studies. To test this approach, we mutated four surface residues of the pentameric Mycobacterium tuberculosis mechanosensitive channel of large conductance (MscL) to cysteine residues as anchors for amphiphile attachment. A series of modified ion channels with four amphiphile groups attached per channel subunit was prepared. One construct showed the highest water solubility to a concentration of up to 4mg/ml in the absence of detergent. This analog also formed native-like, alpha-helical homo-pentamers in the absence of detergent as judged by circular dichroism spectroscopy, size-exclusion chromatography and various light-scattering techniques. Proteins with longer, or shorter polymers attached, or proteins modified exclusively with polar cysteine-reactive small molecules, exhibited reduced to no solubility and higher-order aggregation. Electron microscopy revealed a homogeneous population of particles consistent with a pentameric channel. Solubilization of membrane proteins by covalent attachment of amphiphiles results in homogeneous particles that may prove useful for crystallization, solution NMR spectroscopy, and electron microscopy.
Gryphon Therapeutics, 600 Gateway Blvd., South San Francisco, CA 94080, USA.
Christian F W
CF
Becker
Pavel
P
Strop
Randal B
RB
Bass
Kirk C
KC
Hansen
Kaspar P
KP
Locher
Gang
G
Ren
Mark
M
Yeager
Douglas C
DC
Rees
Gerd G
GG
Kochendoerfer
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008854:Microscopy, Electron; D008958:Models, Molecular; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000737:chemistry; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013501:Surface-Active Agents / Q000737:chemistry*; D014867:Water / Q000737:chemistry*
D001426:Bacterial Proteins; D007473:Ion Channels; D021122:Protein Subunits; D013501:Surface-Active Agents; D014867:Water