Sequence-specific {sup 1}H, {sup 13}C, and {sup 15}N resonance assignments for intestinal fatty-acid-binding protein complexed with palmitate (15.4 kDA)
- Washington Univ. School of Medicine, St. Louis, MO (United States)
Intestinal fatty-acid-binding protein (I-FABP) belongs to a family of soluble, cytoplasmic proteins that are thought to function in the intracellular transport and trafficking of polar lipids. Individual members of this protein family have distinct specificities and affinities for fatty acids, cholesterol, bile salts, and retinoids. We are comparing several retinol- and fatty-acid-binding proteins from intestine in order to define the factors that control molecular recognition in this family of proteins. We have established sequential resonance assignments for uniformly {sup 13}C/{sup 15}N-enriched I-FABP complexed with perdeuterated palmitate at pH7.2 and 37{degrees}C. The assignment strategy was similar to that introduced for calmodulin. We employed seven three-dimensional NMR experiments to establish scalar couplings between backbone and sidechain atoms. Backbone atoms were correlated using triple-resonance HNCO, HNCA, TOCSY-HMQC, HCACO, and HCA(CO)N experiments. Sidechain atoms were correlated using CC-TOCSY, HCCH-TOCSY, and TOCSY-HMQC. The correlations of peaks between three-dimensional spectra were established in a computer-assisted manner using NMR COMPASS (Molecular Simulations, Inc.) Using this approach, {sup 1}H, {sup 13}C, and {sup 15}N resonance assignments have been established for 120 of the 131 residues of I-FABP. For 18 residues, amide {sup 1}H and {sup 15}N resonances were unobservable, apparently because of the rapid exchange of amide protons with bulk water at pH 7.2. The missing amide protons correspond to distinct amino acid patterns in the protein sequence, which will be discussed. During the assignment process, several sources of ambiguity in spin correlations were observed. To overcome this ambiguity, the additional inter-residue correlations often observed in the HNCA experiment were used as cross-checks for the sequential backbone assignments.
- Research Organization:
- Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
- OSTI ID:
- 83395
- Report Number(s):
- LA-12893-C; CONF-9403228-; ON: DE95012795; TRN: 95:004732-0026
- Resource Relation:
- Conference: Stable isotope applications in biomolecular structure and mechanisms, Santa Fe, NM (United States), 27-31 Mar 1994; Other Information: PBD: Dec 1994; Related Information: Is Part Of Stable isotope applications in biomolecular structure and mechanisms. A meeting to bring together producers and users of stable-isotope-labeled compounds to assess current and future needs; Trewhella, J.; Cross, T.A.; Unkefer, C.J. [eds.]; PB: 382 p.
- Country of Publication:
- United States
- Language:
- English
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