Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Aug 1;89(15):7222–7226. doi: 10.1073/pnas.89.15.7222

Dynamic NMR spectral analysis and protein folding: identification of a highly populated folding intermediate of rat intestinal fatty acid-binding protein by 19F NMR.

I J Ropson 1, C Frieden 1
PMCID: PMC49678  PMID: 1496015

Abstract

The folding of intestinal fatty-acid binding protein has been monitored by 19F NMR after incorporation of 6-fluorotryptophan into the protein. The two resonances resulting from the two tryptophans of this protein showed different dependencies on denaturant concentration. One of the resonances was in slow chemical exchange between two resonance frequencies, native and completely unfolded. The changes for this resonance occurred over a denaturant concentration range identical to that monitored by circular dichroism or fluorescence during unfolding. The other resonance continued to show changes at concentrations of denaturant well above that needed to complete the unfolding transition as monitored by optical techniques. Site directed mutagenesis showed that tryptophan-82 was the residue responsible for the unexpected behavior. We conclude, based on complete line-shape analysis, that there are significant concentrations of one or more intermediates in equilibrium with the native and unfolded forms. The structure of the intermediate(s) is more similar to the completely unfolded form of the protein than to the native structure, since little if any secondary structure is present. Further, these structure(s) persist at high denaturant concentrations and may represent local initiating sites in the folding of this beta-sheet protein.

Full text

PDF
7226

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baum J., Dobson C. M., Evans P. A., Hanley C. Characterization of a partly folded protein by NMR methods: studies on the molten globule state of guinea pig alpha-lactalbumin. Biochemistry. 1989 Jan 10;28(1):7–13. doi: 10.1021/bi00427a002. [DOI] [PubMed] [Google Scholar]
  2. Broadhurst R. W., Dobson C. M., Hore P. J., Radford S. E., Rees M. L. A photochemically induced dynamic nuclear polarization study of denatured states of lysozyme. Biochemistry. 1991 Jan 15;30(2):405–412. doi: 10.1021/bi00216a015. [DOI] [PubMed] [Google Scholar]
  3. Bycroft M., Matouschek A., Kellis J. T., Jr, Serrano L., Fersht A. R. Detection and characterization of a folding intermediate in barnase by NMR. Nature. 1990 Aug 2;346(6283):488–490. doi: 10.1038/346488a0. [DOI] [PubMed] [Google Scholar]
  4. Drapeau G. R., Brammar W. J., Yanofsky C. Amino acid replacements of the glutamic acid residue at position 48 in the tryptophan synthetase A protein of Escherichia coli. J Mol Biol. 1968 Jul 28;35(2):357–367. doi: 10.1016/s0022-2836(68)80030-0. [DOI] [PubMed] [Google Scholar]
  5. Evans P. A., Topping K. D., Woolfson D. N., Dobson C. M. Hydrophobic clustering in nonnative states of a protein: interpretation of chemical shifts in NMR spectra of denatured states of lysozyme. Proteins. 1991;9(4):248–266. doi: 10.1002/prot.340090404. [DOI] [PubMed] [Google Scholar]
  6. Gerig J. T. Fluorine nuclear magnetic resonance of fluorinated ligands. Methods Enzymol. 1989;177:3–23. doi: 10.1016/0076-6879(89)77003-8. [DOI] [PubMed] [Google Scholar]
  7. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  8. Li E., Quian S. J., Nader L., Yang N. C., d'Avignon A., Sacchettini J. C., Gordon J. I. Nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol-binding protein II produced in Escherichia coli. Analysis of the apoprotein and the holoprotein containing bound all-trans-retinol and all-trans-retinal. J Biol Chem. 1989 Oct 15;264(29):17041–17048. [PubMed] [Google Scholar]
  9. Liu X. H., Scott P. G., Otter A., Kotovych G. Solution conformation of the type I collagen alpha-2 chain telopeptides studied by 1H and 13C NMR spectroscopy. J Biomol Struct Dyn. 1990 Aug;8(1):63–80. doi: 10.1080/07391102.1990.10507790. [DOI] [PubMed] [Google Scholar]
  10. Luck L. A., Falke J. J. 19F NMR studies of the D-galactose chemosensory receptor. 1. Sugar binding yields a global structural change. Biochemistry. 1991 Apr 30;30(17):4248–4256. doi: 10.1021/bi00231a021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Miranker A., Radford S. E., Karplus M., Dobson C. M. Demonstration by NMR of folding domains in lysozyme. Nature. 1991 Feb 14;349(6310):633–636. doi: 10.1038/349633a0. [DOI] [PubMed] [Google Scholar]
  12. Otter A., Scott P. G., Liu X. H., Kotovych G. A 1H and 13C NMR study on the role of salt-bridges in the formation of a type I beta-turn in N-acetyl-L-Asp-L-Glu-L-Lys-L-Ser-NH2. J Biomol Struct Dyn. 1989 Dec;7(3):455–476. [PubMed] [Google Scholar]
  13. Rao B. D. Nuclear magnetic resonance line-shape analysis and determination of exchange rates. Methods Enzymol. 1989;176:279–311. doi: 10.1016/0076-6879(89)76016-x. [DOI] [PubMed] [Google Scholar]
  14. Roder H., Elöve G. A., Englander S. W. Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature. 1988 Oct 20;335(6192):700–704. doi: 10.1038/335700a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Roder H. Structural characterization of protein folding intermediates by proton magnetic resonance and hydrogen exchange. Methods Enzymol. 1989;176:446–473. doi: 10.1016/0076-6879(89)76024-9. [DOI] [PubMed] [Google Scholar]
  16. Ropson I. J., Gordon J. I., Frieden C. Folding of a predominantly beta-structure protein: rat intestinal fatty acid binding protein. Biochemistry. 1990 Oct 16;29(41):9591–9599. doi: 10.1021/bi00493a013. [DOI] [PubMed] [Google Scholar]
  17. Sacchettini J. C., Banaszak L. J., Gordon J. I. Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction. 1990 Oct 15-Nov 8Mol Cell Biochem. 98(1-2):81–93. doi: 10.1007/BF00231371. [DOI] [PubMed] [Google Scholar]
  18. Sacchettini J. C., Gordon J. I., Banaszak L. J. Crystal structure of rat intestinal fatty-acid-binding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate. J Mol Biol. 1989 Jul 20;208(2):327–339. doi: 10.1016/0022-2836(89)90392-6. [DOI] [PubMed] [Google Scholar]
  19. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Scapin G., Gordon J. I., Sacchettini J. C. Refinement of the structure of recombinant rat intestinal fatty acid-binding apoprotein at 1.2-A resolution. J Biol Chem. 1992 Feb 25;267(6):4253–4269. doi: 10.2210/pdb1ifc/pdb. [DOI] [PubMed] [Google Scholar]
  21. Shen Q. C., Simplaceanu V., Cottam P. F., Wu J. L., Hong J. S., Ho C. Molecular genetic, biochemical and nuclear magnetic resonance studies on the role of the tryptophan residues of glutamine-binding protein from Escherichia coli. J Mol Biol. 1989 Dec 20;210(4):859–867. doi: 10.1016/0022-2836(89)90113-7. [DOI] [PubMed] [Google Scholar]
  22. Skolnick J., Kolinski A., Yaris R. Monte Carlo simulations of the folding of beta-barrel globular proteins. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5057–5061. doi: 10.1073/pnas.85.14.5057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sumner S. C., Gallagher K. S., Davis D. G., Covell D. G., Jernigan R. L., Ferretti J. A. Conformational analysis of the tachykinins in solution: substance P and physalaemin. J Biomol Struct Dyn. 1990 Dec;8(3):687–707. doi: 10.1080/07391102.1990.10507836. [DOI] [PubMed] [Google Scholar]
  24. Sweetser D. A., Heuckeroth R. O., Gordon J. I. The metabolic significance of mammalian fatty-acid-binding proteins: abundant proteins in search of a function. Annu Rev Nutr. 1987;7:337–359. doi: 10.1146/annurev.nu.07.070187.002005. [DOI] [PubMed] [Google Scholar]
  25. Truong H. T., Pratt E. A., Rule G. S., Hsue P. Y., Ho C. Inactive and temperature-sensitive folding mutants generated by tryptophan substitutions in the membrane-bound d-lactate dehydrogenase of Escherichia coli. Biochemistry. 1991 Nov 5;30(44):10722–10729. doi: 10.1021/bi00108a017. [DOI] [PubMed] [Google Scholar]
  26. Udgaonkar J. B., Baldwin R. L. NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A. Nature. 1988 Oct 20;335(6192):694–699. doi: 10.1038/335694a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES