Analysis of side chain rotational restrictions of membrane-embedded proteins by spin-label ESR spectroscopy.
Site-directed spin-labeling electron spin resonance (SDSL-ESR) is a promising tool for membrane protein structure determination. Here we propose a novel way to translate the local structural constraints gained by SDSL-ESR data into a low-resolution structure of a protein by simulating the restrictions of the local conformational spaces of the spin label attached at different protein sites along the primary structure of the membrane-embedded protein. We test the sensitivity of this approach for membrane-embedded M13 major coat protein decorated with a limited number of strategically placed spin labels employing high-throughput site-directed mutagenesis. We find a reasonably good agreement of the simulated and the experimental data taking a protein conformation close to the one determined by fluorescence resonance energy transfer analysis [P.V. Nazarov, R.B.M. Koehorst, W.L. Vos, V.V. Apanasovich, M.A. Hemminga, FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein, Biophys. J. 92 (2007) 1296-1305].
Strancar
Janez
J
Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Kavalenka
Aleh
A
Ziherl
Primoz
P
Stopar
David
D
Hemminga
Marcus A
MA
D036022:Capsid Proteins / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D013113:Spin Labels
D036022:Capsid Proteins; D008055:Lipids; D008565:Membrane Proteins; D013113:Spin Labels