A 1D sensitivity-enhanced 1H spin diffusion experiment for determining membrane protein topology.
A sensitivity-enhanced 1D (1)H spin diffusion experiment, CHH, for determining membrane protein topology is introduced. By transferring the magnetization of the labeled protein (13)C to lipid and water protons for detection, the CHH experiment reduces the time of the original 2D (13)C-detected experiment by two orders of magnitude. The sensitivity enhancement results from (1)H detection and the elimination of the (13)C dimension. Consideration of the spin statistics of the membrane sample indicates that the CHH sensitivity depends on the (13)C labeling level and the number of protein protons relative to the mobile protons. 5-35% of the theoretical sensitivity was achieved on two extensively (13)C labeled proteins. The experimental uncertainties arise from incomplete suppression of the equilibrium (1)H magnetization and the magnetization of lipid protons directly bonded to natural-abundance carbons. The technique, demonstrated on colicin Ia channel domain, confirms the presence of a transmembrane domain and the predominance of surface-bound helices.
Luo
Wenbin
W
Department of Chemistry, Iowa State University, Gilman 0108, Ames, IA 50011, USA.
Hong
Mei
M
D003087:Colicins / Q000032:analysis* / Q000737:chemistry; D004058:Diffusion; D008051:Lipid Bilayers / Q000032:analysis* / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008560:Membrane Fluidity; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D010743:Phospholipids / Q000032:analysis* / Q000737:chemistry; D011487:Protein Conformation; D011522:Protons; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted; D013113:Spin Labels
D003087:Colicins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010743:Phospholipids; D011522:Protons; D013113:Spin Labels