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. 1996 Jun;5(6):1108–1117. doi: 10.1002/pro.5560050613

Intrinsic tryptophans of CRABPI as probes of structure and folding.

P L Clark 1, Z P Liu 1, J Zhang 1, L M Gierasch 1
PMCID: PMC2143446  PMID: 8762142

Abstract

The native state fluorescence and CD spectra of the predominantly beta-sheet cellular retinoic acid-binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three-dimensional structure. Using a combination of spectroscopic approaches and single Trp-mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence-silent due to its interaction with the guanidino group of Arg 111. Although the far-UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic-charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near-UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near-UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge-aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped-flow fluorescence. These dynamic experiments suggest the early evolution of a well-populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.

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Selected References

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  1. Banaszak L., Winter N., Xu Z., Bernlohr D. A., Cowan S., Jones T. A. Lipid-binding proteins: a family of fatty acid and retinoid transport proteins. Adv Protein Chem. 1994;45:89–151. doi: 10.1016/s0065-3233(08)60639-7. [DOI] [PubMed] [Google Scholar]
  2. Cogan U., Kopelman M., Mokady S., Shinitzky M. Binding affinities of retinol and related compounds to retinol binding proteins. Eur J Biochem. 1976 May 17;65(1):71–78. doi: 10.1111/j.1432-1033.1976.tb10390.x. [DOI] [PubMed] [Google Scholar]
  3. Johnson W. C., Jr Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7(3):205–214. doi: 10.1002/prot.340070302. [DOI] [PubMed] [Google Scholar]
  4. Johnson W. C., Jr Secondary structure of proteins through circular dichroism spectroscopy. Annu Rev Biophys Biophys Chem. 1988;17:145–166. doi: 10.1146/annurev.bb.17.060188.001045. [DOI] [PubMed] [Google Scholar]
  5. Kleywegt G. J., Bergfors T., Senn H., Le Motte P., Gsell B., Shudo K., Jones T. A. Crystal structures of cellular retinoic acid binding proteins I and II in complex with all-trans-retinoic acid and a synthetic retinoid. Structure. 1994 Dec 15;2(12):1241–1258. doi: 10.1016/s0969-2126(94)00125-1. [DOI] [PubMed] [Google Scholar]
  6. Liu Z. P., Rizo J., Gierasch L. M. Equilibrium folding studies of cellular retinoic acid binding protein, a predominantly beta-sheet protein. Biochemistry. 1994 Jan 11;33(1):134–142. doi: 10.1021/bi00167a017. [DOI] [PubMed] [Google Scholar]
  7. Locke B. C., MacInnis J. M., Qian S., Gordon J. I., Li E., Fleming G. R., Yang N. C. Fluorescence studies of rat cellular retinol binding protein II produced in Escherichia coli: an analysis of four tryptophan substitution mutants. Biochemistry. 1992 Mar 3;31(8):2376–2383. doi: 10.1021/bi00123a024. [DOI] [PubMed] [Google Scholar]
  8. Loewenthal R., Sancho J., Fersht A. R. Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence. Biochemistry. 1991 Jul 9;30(27):6775–6779. doi: 10.1021/bi00241a021. [DOI] [PubMed] [Google Scholar]
  9. Rizo J., Liu Z. P., Gierasch L. M. 1H and 15N resonance assignments and secondary structure of cellular retinoic acid-binding protein with and without bound ligand. J Biomol NMR. 1994 Nov;4(6):741–760. doi: 10.1007/BF00398406. [DOI] [PubMed] [Google Scholar]
  10. Ropson I. J., Frieden C. Dynamic NMR spectral analysis and protein folding: identification of a highly populated folding intermediate of rat intestinal fatty acid-binding protein by 19F NMR. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7222–7226. doi: 10.1073/pnas.89.15.7222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Rule G. S., Pratt E. A., Simplaceanu V., Ho C. Nuclear magnetic resonance and molecular genetic studies of the membrane-bound D-lactate dehydrogenase of Escherichia coli. Biochemistry. 1987 Jan 27;26(2):549–556. doi: 10.1021/bi00376a029. [DOI] [PubMed] [Google Scholar]
  13. Strickland E. H. Aromatic contributions to circular dichroism spectra of proteins. CRC Crit Rev Biochem. 1974 Jan;2(1):113–175. doi: 10.3109/10409237409105445. [DOI] [PubMed] [Google Scholar]
  14. Varley P., Gronenborn A. M., Christensen H., Wingfield P. T., Pain R. H., Clore G. M. Kinetics of folding of the all-beta sheet protein interleukin-1 beta. Science. 1993 May 21;260(5111):1110–1113. doi: 10.1126/science.8493553. [DOI] [PubMed] [Google Scholar]
  15. Vuilleumier S., Sancho J., Loewenthal R., Fersht A. R. Circular dichroism studies of barnase and its mutants: characterization of the contribution of aromatic side chains. Biochemistry. 1993 Oct 5;32(39):10303–10313. doi: 10.1021/bi00090a005. [DOI] [PubMed] [Google Scholar]
  16. Wasylewski Z., Kołoczek H., Waśniowska A., Slizowska K. Red-edge excitation fluorescence measurements of several two-tryptophan-containing proteins. Eur J Biochem. 1992 May 15;206(1):235–242. doi: 10.1111/j.1432-1033.1992.tb16921.x. [DOI] [PubMed] [Google Scholar]
  17. Zhang J., Liu Z. P., Jones T. A., Gierasch L. M., Sambrook J. F. Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability. Proteins. 1992 Apr;13(2):87–99. doi: 10.1002/prot.340130202. [DOI] [PubMed] [Google Scholar]

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