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. 1992 Jan;1(1):31–45. doi: 10.1002/pro.5560010105

The importance of surface loops for stabilizing an eightfold beta alpha barrel protein.

R Urfer 1, K Kirschner 1
PMCID: PMC2142075  PMID: 1304881

Abstract

An important step in understanding how a protein folds is to determine those regions of the sequence that are critical to both its stability and its folding pathway. We chose phosphoribosyl anthranilate isomerase from Escherichia coli, which is a monomeric representative of the (beta alpha)8 barrel family of proteins, to construct a variant that carries an internal tandem duplication of the fifth beta alpha module. This (beta alpha)9 variant was enzymically active and therefore must have a wild-type (beta alpha)8 core. It had a choice a priori to fold to three different folding frames, which are distinguished by carrying the duplicated segment as an insert into one out of three different loops. Steady-state kinetic constants, the fluorescence properties of a crucial tryptophan residue, and limited proteolysis showed that the stable (beta alpha)9 variant carries the insertion between beta-strand 5 and alpha-helix 5. This preference can be explained by the important role of loops between alpha helices and beta strands in stabilizing the structure of the enzyme.

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Selected References

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  1. Bisswanger H., Kirschner K., Cohn W., Hager V., Hansson E. N-(5-Phosphoribosyl)anthranilate isomerase-indoleglycerol-phosphate synthase. 1. A substrate analogue binds to two different binding sites on the bifunctional enzyme from Escherichia coli. Biochemistry. 1979 Dec 25;18(26):5946–5953. doi: 10.1021/bi00593a029. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Certa U., Bannwarth W., Stüber D., Gentz R., Lanzer M., Le Grice S., Guillot F., Wendler I., Hunsmann G., Bujard H. Subregions of a conserved part of the HIV gp41 transmembrane protein are differentially recognized by antibodies of infected individuals. EMBO J. 1986 Nov;5(11):3051–3056. doi: 10.1002/j.1460-2075.1986.tb04605.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chaffotte A. F., Goldberg M. E. Fluorescence-quenching studies on a conformational transition within a domain of the beta 2 subunit of Escherichia coli tryptophan synthase. Eur J Biochem. 1984 Feb 15;139(1):47–50. doi: 10.1111/j.1432-1033.1984.tb07974.x. [DOI] [PubMed] [Google Scholar]
  5. Chothia C., Finkelstein A. V. The classification and origins of protein folding patterns. Annu Rev Biochem. 1990;59:1007–1039. doi: 10.1146/annurev.bi.59.070190.005043. [DOI] [PubMed] [Google Scholar]
  6. Chou K. C., Carlacci L. Energetic approach to the folding of alpha/beta barrels. Proteins. 1991;9(4):280–295. doi: 10.1002/prot.340090406. [DOI] [PubMed] [Google Scholar]
  7. Crawford I. P., Clarke M., van Cleemput M., Yanofsky C. Crucial role of the connecting region joining the two functional domains of yeast tryptophan synthetase. J Biol Chem. 1987 Jan 5;262(1):239–244. [PubMed] [Google Scholar]
  8. Eberhard M. A set of programs for analysis of kinetic and equilibrium data. Comput Appl Biosci. 1990 Jul;6(3):213–221. doi: 10.1093/bioinformatics/6.3.213. [DOI] [PubMed] [Google Scholar]
  9. Eftink M. R., Ghiron C. A. Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry. 1976 Feb 10;15(3):672–680. doi: 10.1021/bi00648a035. [DOI] [PubMed] [Google Scholar]
  10. Farber G. K., Petsko G. A. The evolution of alpha/beta barrel enzymes. Trends Biochem Sci. 1990 Jun;15(6):228–234. doi: 10.1016/0968-0004(90)90035-a. [DOI] [PubMed] [Google Scholar]
  11. Fetrow J. S., Cardillo T. S., Sherman F. Deletions and replacements of omega loops in yeast iso-1-cytochrome c. Proteins. 1989;6(4):372–381. doi: 10.1002/prot.340060404. [DOI] [PubMed] [Google Scholar]
  12. Finkelstein A. V., Ptitsyn O. B. Why do globular proteins fit the limited set of folding patterns? Prog Biophys Mol Biol. 1987;50(3):171–190. doi: 10.1016/0079-6107(87)90013-7. [DOI] [PubMed] [Google Scholar]
  13. Hommel U., Lustig A., Kirschner K. Purification and characterization of yeast anthranilate phosphoribosyltransferase. Eur J Biochem. 1989 Mar 1;180(1):33–40. doi: 10.1111/j.1432-1033.1989.tb14611.x. [DOI] [PubMed] [Google Scholar]
  14. Hynes T. R., Kautz R. A., Goodman M. A., Gill J. F., Fox R. O. Transfer of a beta-turn structure to a new protein context. Nature. 1989 May 4;339(6219):73–76. doi: 10.1038/339073a0. [DOI] [PubMed] [Google Scholar]
  15. Jaenicke R. Protein folding: local structures, domains, subunits, and assemblies. Biochemistry. 1991 Apr 2;30(13):3147–3161. doi: 10.1021/bi00227a001. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lesk A. M., Brändén C. I., Chothia C. Structural principles of alpha/beta barrel proteins: the packing of the interior of the sheet. Proteins. 1989;5(2):139–148. doi: 10.1002/prot.340050208. [DOI] [PubMed] [Google Scholar]
  18. Leszczynski J. F., Rose G. D. Loops in globular proteins: a novel category of secondary structure. Science. 1986 Nov 14;234(4778):849–855. doi: 10.1126/science.3775366. [DOI] [PubMed] [Google Scholar]
  19. Lim W. A., Sauer R. T. The role of internal packing interactions in determining the structure and stability of a protein. J Mol Biol. 1991 May 20;219(2):359–376. doi: 10.1016/0022-2836(91)90570-v. [DOI] [PubMed] [Google Scholar]
  20. Lindqvist Y. Refined structure of spinach glycolate oxidase at 2 A resolution. J Mol Biol. 1989 Sep 5;209(1):151–166. doi: 10.1016/0022-2836(89)90178-2. [DOI] [PubMed] [Google Scholar]
  21. Mollet B., Delley M. A beta-galactosidase deletion mutant of Lactobacillus bulgaricus reverts to generate an active enzyme by internal DNA sequence duplication. Mol Gen Genet. 1991 May;227(1):17–21. doi: 10.1007/BF00260700. [DOI] [PubMed] [Google Scholar]
  22. Rice P. A., Goldman A., Steitz T. A. A helix-turn-strand structural motif common in alpha-beta proteins. Proteins. 1990;8(4):334–340. doi: 10.1002/prot.340080407. [DOI] [PubMed] [Google Scholar]
  23. Riechmann L., Clark M., Waldmann H., Winter G. Reshaping human antibodies for therapy. Nature. 1988 Mar 24;332(6162):323–327. doi: 10.1038/332323a0. [DOI] [PubMed] [Google Scholar]
  24. Schneider W. P., Nichols B. P., Yanofsky C. Procedure for production of hybrid genes and proteins and its use in assessing significance of amino acid differences in homologous tryptophan synthetase alpha polypeptides. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2169–2173. doi: 10.1073/pnas.78.4.2169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stanssens P., Opsomer C., McKeown Y. M., Kramer W., Zabeau M., Fritz H. J. Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res. 1989 Jun 26;17(12):4441–4454. doi: 10.1093/nar/17.12.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thornton J. M., Sibanda B. L., Edwards M. S., Barlow D. J. Analysis, design and modification of loop regions in proteins. Bioessays. 1988 Feb-Mar;8(2):63–69. doi: 10.1002/bies.950080205. [DOI] [PubMed] [Google Scholar]
  27. Tramontano A., Chothia C., Lesk A. M. Structural determinants of the conformations of medium-sized loops in proteins. Proteins. 1989;6(4):382–394. doi: 10.1002/prot.340060405. [DOI] [PubMed] [Google Scholar]
  28. Wilmanns M., Hyde C. C., Davies D. R., Kirschner K., Jansonius J. N. Structural conservation in parallel beta/alpha-barrel enzymes that catalyze three sequential reactions in the pathway of tryptophan biosynthesis. Biochemistry. 1991 Sep 24;30(38):9161–9169. doi: 10.1021/bi00102a006. [DOI] [PubMed] [Google Scholar]

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