H-1, C-13 AND N-15 ASSIGNMENTS AND CHEMICAL SHIFT-DERIVED SECONDARY STRUCTURE OF INTESTINAL FATTY-ACID-BINDING PROTEIN

被引:21
作者
HODSDON, ME [1 ]
TONER, JJ [1 ]
CISTOLA, DP [1 ]
机构
[1] ST LOUIS UNIV,SCH MED,DEPT BIOCHEM & MOLEC BIOPHYS,ST LOUIS,MO 63110
关键词
TRIPLE-RESONANCE 3D NMR; LIPID-BINDING PROTEINS; ISOTOPE ENRICHMENTS; RESONANCE ASSIGNMENTS; CHEMICAL SHIFT; PROTEIN SECONDARY STRUCTURE;
D O I
10.1007/BF00211784
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Sequence-specific H-1, C-13 and N-15 resonance assignments have been established for rat intestinal fatty acid-binding protein complexed with palmitate (15.4 kDa) at pH 7.2 and 37 degrees C. The resonance assignment strategy involved the concerted use of seven 3D triple-resonance experiments (CC-TOCSY, HCCH-TOCSY, HNCO, HNCA, N-15-TOCSY-HMQC, HCACO and HCA(CO)N). A central feature of this strategy was the concurrent assignment of both backbone and side-chain aliphatic atoms, which was critical for overcoming ambiguities in the assignment process. The CC-TOCSY experiment provided the unambiguous links between the side-chain spin systems observed in HCCH-TOCSY and the backbone correlations observed in the other experiments. Assignments were established for 124 of the 131 residues, although 6 of the 124 had missing amide H-1 resonances, presumably due to rapid-exchange with solvent under these experimental conditions. The assignment database was used to determine the solution secondary structure of the complex, based on chemical shift indices for the H-1(alpha), C-13(alpha), C-13(beta), and (CO)-C-13 atoms. Overall, the secondary structure agreed well with that determined by X-ray crystallography [Sacchettini et al. (1989) J. Mol. Biol., 208, 327-339], although minor differences were observed at the edges of secondary structure elements.
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页码:198 / 210
页数:13
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