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. 1992 Jan 1;281(Pt 1):67–72. doi: 10.1042/bj2810067

Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae.

S Craig 1, J M Clements 1, A L Cook 1, D T Dryden 1, D R Green 1, K Heremans 1, P M Kirwin 1, M J Price 1, A Fallon 1
PMCID: PMC1130641  PMID: 1731769

Abstract

A detailed biophysical study of the secondary and tertiary structures of recombinant platelet-derived growth factor (PDGF)-BB produced in yeast has been carried out. The secondary structure of the molecule is composed of 54% beta-sheet with less than 5% ordered helix. The single tryptophan residue has been shown to be solvent-accessible; however, the ability of the side chain to rotate is severely restricted. The fluorescence emission is quenched at pH 7.0 and in the presence of high salt, but dequenched by titration to lower pH with a pK of 5.8. Two proteinase-resistant mutants of PDGF [( Ser28]- and [Pro32]-PDGF-BB) have also been characterized and shown to have secondary and tertiary structures indistinguishable from wild-type PDGF-BB. These are, therefore, suitable stable background molecules in which to carry out structure-activity-relationship studies on PDGF-BB.

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Selected References

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  1. Cook A. L., Kirwin P. M., Craig S., Bawden L. J., Green D. R., Price M. J., Richardson S. J., Fallon A., Drummond A. H., Edwards R. M. Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity. Biochem J. 1992 Jan 1;281(Pt 1):57–65. doi: 10.1042/bj2810057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Craig S., Hollecker M., Creighton T. E., Pain R. H. Single amino acid mutations block a late step in the folding of beta-lactamase from Staphylococcus aureus. J Mol Biol. 1985 Oct 20;185(4):681–687. doi: 10.1016/0022-2836(85)90053-1. [DOI] [PubMed] [Google Scholar]
  3. Craig S., Schmeissner U., Wingfield P., Pain R. H. Conformation, stability, and folding of interleukin 1 beta. Biochemistry. 1987 Jun 16;26(12):3570–3576. doi: 10.1021/bi00386a048. [DOI] [PubMed] [Google Scholar]
  4. Hoppe J., Weich H. A., Eichner W. Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli. Biochemistry. 1989 Apr 4;28(7):2956–2960. doi: 10.1021/bi00433a032. [DOI] [PubMed] [Google Scholar]
  5. Hoppe J., Weich H. A., Eichner W., Tatje D. Preparation of biologically active platelet-derived growth factor isoforms AA and AB. Preferential formation of AB heterodimers. Eur J Biochem. 1990 Jan 12;187(1):207–214. doi: 10.1111/j.1432-1033.1990.tb15296.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Lehrer S. S. Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry. 1971 Aug 17;10(17):3254–3263. doi: 10.1021/bi00793a015. [DOI] [PubMed] [Google Scholar]
  8. Levitt M., Greer J. Automatic identification of secondary structure in globular proteins. J Mol Biol. 1977 Aug 5;114(2):181–239. doi: 10.1016/0022-2836(77)90207-8. [DOI] [PubMed] [Google Scholar]
  9. Parry D. A. Coiled-coils in alpha-helix-containing proteins: analysis of the residue types within the heptad repeat and the use of these data in the prediction of coiled-coils in other proteins. Biosci Rep. 1982 Dec;2(12):1017–1024. doi: 10.1007/BF01122170. [DOI] [PubMed] [Google Scholar]
  10. Provencher S. W., Glöckner J. Estimation of globular protein secondary structure from circular dichroism. Biochemistry. 1981 Jan 6;20(1):33–37. doi: 10.1021/bi00504a006. [DOI] [PubMed] [Google Scholar]
  11. Ross R., Raines E. W., Bowen-Pope D. F. The biology of platelet-derived growth factor. Cell. 1986 Jul 18;46(2):155–169. doi: 10.1016/0092-8674(86)90733-6. [DOI] [PubMed] [Google Scholar]
  12. Settineri C. A., Medzihradszky K. F., Masiarz F. R., Burlingame A. L., Chu C., George-Nascimento C. Characterization of O-glycosylation sites in recombinant B-chain of platelet-derived growth factor expressed in yeast using liquid secondary ion mass spectrometry, tandem mass spectrometry and Edman sequence analysis. Biomed Environ Mass Spectrom. 1990 Nov;19(11):665–676. doi: 10.1002/bms.1200191106. [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. Vogel S., Hoppe J. Binding domains and epitopes in platelet-derived growth factor. Biochemistry. 1989 Apr 4;28(7):2961–2966. doi: 10.1021/bi00433a033. [DOI] [PubMed] [Google Scholar]
  15. Williams R. W. Estimation of protein secondary structure from the laser Raman amide I spectrum. J Mol Biol. 1983 Jun 5;166(4):581–603. doi: 10.1016/s0022-2836(83)80285-x. [DOI] [PubMed] [Google Scholar]
  16. Williams R. W. Protein secondary structure analysis using Raman amide I and amide III spectra. Methods Enzymol. 1986;130:311–331. doi: 10.1016/0076-6879(86)30016-8. [DOI] [PubMed] [Google Scholar]

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