Amino-acid selective experiments on uniformly 13C and 15N labeled proteins by MAS NMR: Filtering of lysines and arginines.
Amino-acid selective magic-angle spinning (MAS) NMR experiments can aid the assignment of ambiguous cross-peaks in crowded spectra of solid proteins. In particular for larger proteins, data analysis can be hindered by severe resonance overlap. In such cases, filtering techniques may provide a good alternative to site-specific spin-labeling to obtain unambiguous assignments that can serve as starting points in the assignment procedure. In this paper we present a simple pulse sequence that allows selective excitation of arginine and lysine residues. To achieve this, we make use of a combination of specific cross-polarization for selective excitation [M. Baldus, A.T. Petkova, J. Herzfeld, R.G. Griffin, Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems, Mol. Phys. 95 (1998) 1197-1207.] and spin diffusion for transfer along the amino-acid side-chain. The selectivity of the filter is demonstrated with the excitation of lysine and arginine side-chain resonances in a uniformly 13C and 15N labeled protein preparation of the alpha-spectrin SH3 domain. It is shown that the filter can be applied as a building block in a 13C-13C lysine-only correlation experiment.
Jehle
Stefan
S
Leibniz-Institut für molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, D-13125 Berlin, Germany.
Rehbein
Kristina
K
Diehl
Anne
A
van Rossum
Barth-Jan
BJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001120:Arginine / Q000032:analysis*; D001923:Brain Chemistry; D002247:Carbon Isotopes / Q000032:analysis / Q000737:chemistry; D002645:Chickens; D007553:Isotope Labeling / Q000379:methods; D008239:Lysine / Q000032:analysis*; D009587:Nitrogen Isotopes / Q000032:analysis / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013113:Spin Labels
D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D013113:Spin Labels; D001120:Arginine; D008239:Lysine