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. 1995 Apr;4(4):613–621. doi: 10.1002/pro.5560040403

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

J A Huntington 1, P A Patston 1, P G Gettins 1
PMCID: PMC2143095  PMID: 7613461

Abstract

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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  1. Batra P. P., Roebuck M. A., Uetrecht D. Effect of lysine modification on the secondary structure of ovalbumin. J Protein Chem. 1990 Feb;9(1):37–44. doi: 10.1007/BF01024982. [DOI] [PubMed] [Google Scholar]
  2. Bode W., Papamokos E., Musil D., Seemueller U., Fritz H. Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin. EMBO J. 1986 Apr;5(4):813–818. doi: 10.1002/j.1460-2075.1986.tb04286.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bode W., Papamokos E., Musil D. The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Structural analysis, subtilisin structure and interface geometry. Eur J Biochem. 1987 Aug 3;166(3):673–692. doi: 10.1111/j.1432-1033.1987.tb13566.x. [DOI] [PubMed] [Google Scholar]
  4. Carrell R. W., Evans D. L., Stein P. E. Mobile reactive centre of serpins and the control of thrombosis. Nature. 1991 Oct 10;353(6344):576–578. doi: 10.1038/353576a0. [DOI] [PubMed] [Google Scholar]
  5. Gettins P. Absence of large-scale conformational change upon limited proteolysis of ovalbumin, the prototypic serpin. J Biol Chem. 1989 Mar 5;264(7):3781–3785. [PubMed] [Google Scholar]
  6. Gettins P., Patston P. A., Schapira M. The role of conformational change in serpin structure and function. Bioessays. 1993 Jul;15(7):461–467. doi: 10.1002/bies.950150705. [DOI] [PubMed] [Google Scholar]
  7. Hood D. B., Huntington J. A., Gettins P. G. Alpha 1-proteinase inhibitor variant T345R. Influence of P14 residue on substrate and inhibitory pathways. Biochemistry. 1994 Jul 19;33(28):8538–8547. doi: 10.1021/bi00194a020. [DOI] [PubMed] [Google Scholar]
  8. Hopkins P. C., Carrell R. W., Stone S. R. Effects of mutations in the hinge region of serpins. Biochemistry. 1993 Aug 3;32(30):7650–7657. doi: 10.1021/bi00081a008. [DOI] [PubMed] [Google Scholar]
  9. Johnson B. A., Shirokawa J. M., Aswad D. W. Deamidation of calmodulin at neutral and alkaline pH: quantitative relationships between ammonia loss and the susceptibility of calmodulin to modification by protein carboxyl methyltransferase. Arch Biochem Biophys. 1989 Jan;268(1):276–286. doi: 10.1016/0003-9861(89)90589-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Lawrence D. A., Olson S. T., Palaniappan S., Ginsburg D. Engineering plasminogen activator inhibitor 1 mutants with increased functional stability. Biochemistry. 1994 Mar 29;33(12):3643–3648. doi: 10.1021/bi00178a022. [DOI] [PubMed] [Google Scholar]
  12. Lawrence D. A., Olson S. T., Palaniappan S., Ginsburg D. Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition. J Biol Chem. 1994 Nov 4;269(44):27657–27662. [PubMed] [Google Scholar]
  13. Manavalan P., Johnson W. C., Jr Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal Biochem. 1987 Nov 15;167(1):76–85. doi: 10.1016/0003-2697(87)90135-7. [DOI] [PubMed] [Google Scholar]
  14. Munch M., Heegaard C., Jensen P. H., Andreasen P. A. Type-1 inhibitor of plasminogen activators. Distinction between latent, activated and reactive centre-cleaved forms with thermal stability and monoclonal antibodies. FEBS Lett. 1991 Dec 16;295(1-3):102–106. doi: 10.1016/0014-5793(91)81395-o. [DOI] [PubMed] [Google Scholar]
  15. OTTESEN M. The transformation of ovalbumin into plakalbumin; a case of limited proteolysis. C R Trav Lab Carlsberg Chim. 1958;30(14):211–270. [PubMed] [Google Scholar]
  16. OTTESEN M., WOLLENBERGER A. Stepwise degradation of the peptides liberated in the transformation of ovalbumin to plakalbumin. C R Trav Lab Carlsberg Chim. 1953;28(18):463–476. [PubMed] [Google Scholar]
  17. Olson S. T. Heparin and ionic strength-dependent conversion of antithrombin III from an inhibitor to a substrate of alpha-thrombin. J Biol Chem. 1985 Aug 25;260(18):10153–10160. [PubMed] [Google Scholar]
  18. Patston P. A., Gettins P., Beechem J., Schapira M. Mechanism of serpin action: evidence that C1 inhibitor functions as a suicide substrate. Biochemistry. 1991 Sep 10;30(36):8876–8882. doi: 10.1021/bi00100a022. [DOI] [PubMed] [Google Scholar]
  19. Privalov P. L. Stability of proteins. Proteins which do not present a single cooperative system. Adv Protein Chem. 1982;35:1–104. [PubMed] [Google Scholar]
  20. Rubin H., Wang Z. M., Nickbarg E. B., McLarney S., Naidoo N., Schoenberger O. L., Johnson J. L., Cooperman B. S. Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins. J Biol Chem. 1990 Jan 15;265(2):1199–1207. [PubMed] [Google Scholar]
  21. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  22. Schechter N. M., Sprows J. L., Schoenberger O. L., Lazarus G. S., Cooperman B. S., Rubin H. Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors. J Biol Chem. 1989 Dec 15;264(35):21308–21315. [PubMed] [Google Scholar]
  23. Stein P. E., Leslie A. G., Finch J. T., Carrell R. W. Crystal structure of uncleaved ovalbumin at 1.95 A resolution. J Mol Biol. 1991 Oct 5;221(3):941–959. doi: 10.1016/0022-2836(91)80185-w. [DOI] [PubMed] [Google Scholar]
  24. Stein P. E., Tewkesbury D. A., Carrell R. W. Ovalbumin and angiotensinogen lack serpin S-R conformational change. Biochem J. 1989 Aug 15;262(1):103–107. doi: 10.1042/bj2620103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vogel H. J., Bridger W. A. Phosphorus-31 nuclear magnetic resonance studies of the two phosphoserine residues of hen egg white ovalbumin. Biochemistry. 1982 Nov 9;21(23):5825–5831. doi: 10.1021/bi00266a016. [DOI] [PubMed] [Google Scholar]
  26. Wiman B., Almquist A., Sigurdardottir O., Lindahl T. Plasminogen activator inhibitor 1 (PAI) is bound to vitronectin in plasma. FEBS Lett. 1988 Dec 19;242(1):125–128. doi: 10.1016/0014-5793(88)80999-2. [DOI] [PubMed] [Google Scholar]
  27. Wright H. T., Qian H. X., Huber R. Crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin. Its relationship to the structure of cleaved alpha-1-proteinase inhibitor. J Mol Biol. 1990 Jun 5;213(3):513–528. doi: 10.1016/s0022-2836(05)80212-8. [DOI] [PubMed] [Google Scholar]

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