Sample optimization and identification of signal patterns of amino acid side chains in 2D RFDR spectra of the alpha-spectrin SH3 domain.
Future structural investigations of proteins by solid-state CPMAS NMR will rely on uniformly labeled protein samples showing spectra with an excellent resolution. NMR samples of the solid alpha-spectrin SH3 domain were generated in four different ways, and their (13)C CPMAS spectra were compared. The spectrum of a [u-(13)C, (15)N]-labeled sample generated by precipitation shows very narrow (13)C signals and resolved scalar carbon-carbon couplings. Linewidths of 16-19 Hz were found for the three alanine C(beta )signals of a selectively labeled [70% 3-(13)C]alanine-enriched SH3 sample. The signal pattern of the isoleucine, of all prolines, valines, alanines, and serines, and of three of the four threonines were identified in 2D (13)C-(13)C RFDR spectra of the [u-(13)C, (15)N]-labeled SH3 sample. A comparison of the (13)C chemical shifts of the found signal patterns with the (13)C assignment obtained in solution shows an intriguing match.
Pauli
J
J
Forschungsinstitut für Molekulare Pharmakologie, Alfred-Kowalke-Strasse 4, Berlin, D-10315, Germany.
van Rossum
B
B
Förster
H
H
de Groot
H J
HJ
Oschkinat
H
H
D000409:Alanine / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry*; D002247:Carbon Isotopes; D011232:Chemical Precipitation; D007532:Isoleucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry; D013049:Spectrin / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry; D014633:Valine / Q000737:chemistry; D018909:src Homology Domains
D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D007532:Isoleucine; D013049:Spectrin; D013912:Threonine; D012694:Serine; D011392:Proline; D014633:Valine; D000409:Alanine