Membrane hydrocarbon thickness modulates the dynamics of a membrane transport protein.
Nitroxide spin labels were incorporated into selected sites within the beta-barrel of the bacterial outer-membrane transport protein BtuB by site-directed mutagenesis, followed by chemical modification with a methanethiosufonate spin label. The electron paramagnetic resonance lineshapes of the spin-labeled side chain (R1) from these sites are highly variable, and have spectral parameters that reflect secondary structure and local steric constraints. In addition, these lineshape parameters correlate with crystallographic structure factors for Calpha carbons, suggesting that the motion of the spin label is modulated by both the local modes of motion of the spin label and the local dynamics of the protein backbone. Experiments performed as a function of lipid composition and sample temperature indicate that nitroxide spin labels on the exterior surface of BtuB, which face the membrane hydrocarbon, are not strongly influenced by the phase state of the bulk lipids. However, these spectra are modulated by membrane hydrocarbon thickness. Specifically, the values of the scaled mobility parameter for the R1 lineshapes are inversely proportional to the hydrocarbon thickness. These data suggest that protein dynamics and structure in BtuB are directly coupled to membrane hydrophobic thickness.
Xu
Qi
Q
Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, Virginia 22904-4319, USA.
Kim
Miyeon
M
Ho
K W David
KW
Lachowicz
Pawel
P
Fanucci
Gail E
GE
Cafiso
David S
DS
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006838:Hydrocarbons / Q000737:chemistry*; D016014:Linear Models; D008051:Lipid Bilayers / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009068:Movement; D009589:Nitrogen Oxides / Q000378:metabolism; D017433:Protein Structure, Secondary; D013113:Spin Labels
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D006838:Hydrocarbons; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D009589:Nitrogen Oxides; D013113:Spin Labels; C039900:nitroxyl