Sample Optimization and Identification of Signal Patterns of Amino Acid Side Chains in 2D RFDR Spectra of the α-Spectrin SH3 Domain |
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Authors: | Jutta Pauli Barth van Rossum Hans Frster Huub J M de Groot Hartmut Oschkinat |
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Institution: | a Forschungsinstitut für Molekulare Pharmakologie, Alfred-Kowalke-Strasse 4, D-10315, Berlin, Germany;b Gorlaeus Laboratories, Einsteinweg 55, P.O. Box 9502, 2300, RA Leiden, The Netherlands;c Bruker Analytik GmbH, Am Silberstreifen, Rheinstetten, Germany |
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Abstract: | 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 α-spectrin SH3 domain were generated in four different ways, and their 13C CPMAS spectra were compared. The spectrum of a u-13C, 15N]-labeled sample generated by precipitation shows very narrow 13C signals and resolved scalar carbon–carbon couplings. Linewidths of 16–19 Hz were found for the three alanine Cβ signals of a selectively labeled 70% 3-13C]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 13C–13C RFDR spectra of the u-13C,15N]-labeled SH3 sample. A comparison of the 13C chemical shifts of the found signal patterns with the 13C assignment obtained in solution shows an intriguing match. |
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Keywords: | solid-state NMR spectroscopy protein structure determination resolution RFDR spectroscopy 13C linewidth high magnetic fields |
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