Membrane protein topology probed by (1)H spin diffusion from lipids using solid-state NMR spectroscopy.
We describe a two-dimensional solid-state NMR technique to investigate membrane protein topology under magic-angle spinning conditions. The experiment detects the rate of (1)H spin diffusion from the mobile lipids to the rigid protein. While spin diffusion within the rigid protein is fast, magnetization transfer in the mobile lipids is an inefficient and slow process. Qualitative analysis of (1)H spin-diffusion build-up curves from the lipid chain-end methyl groups to the protein allows the identification of membrane-embedded domains in the protein. Numerical simulations of spin-diffusion build-up curves yield the approximate insertion depth of protein segments in the membrane. The experiment is demonstrated on the selectively (13)C labeled colicin Ia channel domain, known to have a membrane-embedded domain, and on DNA/cationic lipid complexes where the DNA rods are bound to the membrane surface. The experiment is designed for X-nucleus detection, which could be (13)C or (15)N in the protein and (31)P for the DNA. Finally, we show that a qualitative distinction between membrane proteins with and without a membrane-embedded domain can be made even by using an unlabeled protein, by detection of lipid signals. This spin-diffusion experiment is simple to perform and requires no oriented bilayer preparations and only standard NMR hardware.
Huster
Daniel
D
Department of Chemistry, Iowa State University, Gilman Hall 0108, Ames, Iowa 50011, USA.
Yao
Xiaolan
X
Hong
Mei
M
D002247:Carbon Isotopes; D002462:Cell Membrane / Q000737:chemistry; D003087:Colicins / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004058:Diffusion; D008051:Lipid Bilayers / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D011522:Protons
D002247:Carbon Isotopes; D003087:Colicins; D008051:Lipid Bilayers; D008563:Membrane Lipids; D008565:Membrane Proteins; D011522:Protons; D004247:DNA