Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1994 Mar;3(3):411–418. doi: 10.1002/pro.5560030305

Autonomous folding of the excised coenzyme-binding domain of D-glyceraldehyde 3-phosphate dehydrogenase from Thermotoga maritima.

M Jecht 1, A Tomschy 1, K Kirschner 1, R Jaenicke 1
PMCID: PMC2142700  PMID: 8019412

Abstract

An important question in protein folding is whether compact substructures or domains are autonomous units of folding and assembly. The protomer of the tetrameric D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has a complex coenzyme-binding domain, in which residues 1-146 form a compact substructure with the last 31 residues (313-333). Here it is shown that the gene of a single-chain protein can be expressed in Escherichia coli after deleting the 163 codons corresponding to the interspersed catalytic domain (150-312). The purified gene product is a soluble, monomeric protein that binds both NAD+ and NADH strongly and possesses the same unfolding transition induced by guanidinium chloride as the native tetramer. The autonomous folding of the coenzyme-binding domain has interesting implications for the folding, assembly, function, and evolution of the native enzyme.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Adachi O., Kohn L. D., Miles E. W. Crystalline alpha2 beta2 complexes of tryptophan synthetase of Escherichia coli. A comparison between the native complex and the reconstituted complex. J Biol Chem. 1974 Dec 25;249(24):7756–7763. [PubMed] [Google Scholar]
  2. Biesecker G., Harris J. I., Thierry J. C., Walker J. E., Wonacott A. J. Sequence and structure of D-glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus. Nature. 1977 Mar 24;266(5600):328–333. doi: 10.1038/266328a0. [DOI] [PubMed] [Google Scholar]
  3. Bird R. E., Hardman K. D., Jacobson J. W., Johnson S., Kaufman B. M., Lee S. M., Lee T., Pope S. H., Riordan G. S., Whitlow M. Single-chain antigen-binding proteins. Science. 1988 Oct 21;242(4877):423–426. doi: 10.1126/science.3140379. [DOI] [PubMed] [Google Scholar]
  4. Branlant C., Oster T., Branlant G. Nucleotide sequence determination of the DNA region coding for Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase and of the flanking DNA regions required for its expression in Escherichia coli. Gene. 1989 Jan 30;75(1):145–155. doi: 10.1016/0378-1119(89)90391-0. [DOI] [PubMed] [Google Scholar]
  5. Brändeén C. I. Relation between structure and function of alpha/beta-proteins. Q Rev Biophys. 1980 Aug;13(3):317–338. doi: 10.1017/s0033583500001712. [DOI] [PubMed] [Google Scholar]
  6. Böhm G., Muhr R., Jaenicke R. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng. 1992 Apr;5(3):191–195. doi: 10.1093/protein/5.3.191. [DOI] [PubMed] [Google Scholar]
  7. Corbier C., Mougin A., Mely Y., Adolph H. W., Zeppezauer M., Gerard D., Wonacott A., Branlant G. The nicotinamide subsite of glyceraldehyde-3-phosphate dehydrogenase studied by site-directed mutagenesis. Biochimie. 1990 Aug;72(8):545–554. doi: 10.1016/0300-9084(90)90119-2. [DOI] [PubMed] [Google Scholar]
  8. Fling S. P., Gregerson D. S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem. 1986 May 15;155(1):83–88. doi: 10.1016/0003-2697(86)90228-9. [DOI] [PubMed] [Google Scholar]
  9. Griffith J. P., Lee B., Murdock A. L., Amelunxen R. E. Molecular symmetry of glyceraldehyde-3-phosphate dehydrogenase from Bacillus coagulans. J Mol Biol. 1983 Oct 5;169(4):963–974. doi: 10.1016/s0022-2836(83)80145-4. [DOI] [PubMed] [Google Scholar]
  10. Herold M., Leistler B., Hage A., Luger K., Kirschner K. Autonomous folding and coenzyme binding of the excised pyridoxal 5'-phosphate binding domain of aspartate aminotransferase from Escherichia coli. Biochemistry. 1991 Apr 16;30(15):3612–3620. doi: 10.1021/bi00229a004. [DOI] [PubMed] [Google Scholar]
  11. Huston J. S., Levinson D., Mudgett-Hunter M., Tai M. S., Novotný J., Margolies M. N., Ridge R. J., Bruccoleri R. E., Haber E., Crea R. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879–5883. doi: 10.1073/pnas.85.16.5879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jaenicke R. Protein stability and molecular adaptation to extreme conditions. Eur J Biochem. 1991 Dec 18;202(3):715–728. doi: 10.1111/j.1432-1033.1991.tb16426.x. [DOI] [PubMed] [Google Scholar]
  13. Jaenicke R., Vogel W., Rudolph R. Dimeric intermediates in the dissociation of lactic dehydrogenase. Eur J Biochem. 1981 Mar;114(3):525–531. doi: 10.1111/j.1432-1033.1981.tb05176.x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  16. Opitz U., Rudolph R., Jaenicke R., Ericsson L., Neurath H. Proteolytic dimers of porcine muscle lactate dehydrogenase: characterization, folding, and reconstitution of the truncated and nicked polypeptide chain. Biochemistry. 1987 Mar 10;26(5):1399–1406. doi: 10.1021/bi00379a028. [DOI] [PubMed] [Google Scholar]
  17. Rehaber V., Jaenicke R. Stability and reconstitution of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima. J Biol Chem. 1992 Jun 5;267(16):10999–11006. [PubMed] [Google Scholar]
  18. Rehaber V., Jaenicke R. The low-temperature folding intermediate of hyperthermophilic D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima shows a native-like cooperative unfolding transition. FEBS Lett. 1993 Feb 8;317(1-2):163–166. doi: 10.1016/0014-5793(93)81514-z. [DOI] [PubMed] [Google Scholar]
  19. Rossmann M. G., Moras D., Olsen K. W. Chemical and biological evolution of nucleotide-binding protein. Nature. 1974 Jul 19;250(463):194–199. doi: 10.1038/250194a0. [DOI] [PubMed] [Google Scholar]
  20. Schultes V., Deutzmann R., Jaenicke R. Complete amino-acid sequence of glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima. Eur J Biochem. 1990 Aug 28;192(1):25–31. doi: 10.1111/j.1432-1033.1990.tb19190.x. [DOI] [PubMed] [Google Scholar]
  21. Skarzyński T., Moody P. C., Wonacott A. J. Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution. J Mol Biol. 1987 Jan 5;193(1):171–187. doi: 10.1016/0022-2836(87)90635-8. [DOI] [PubMed] [Google Scholar]
  22. Skarzyński T., Wonacott A. J. Coenzyme-induced conformational changes in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. J Mol Biol. 1988 Oct 20;203(4):1097–1118. doi: 10.1016/0022-2836(88)90130-1. [DOI] [PubMed] [Google Scholar]
  23. Stinson R. A., Holbrook J. J. Equilibrium binding of nicotinamide nucleotides to lactate dehydrogenases. Biochem J. 1973 Apr;131(4):719–728. doi: 10.1042/bj1310719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thornton J. M., Sibanda B. L. Amino and carboxy-terminal regions in globular proteins. J Mol Biol. 1983 Jun 25;167(2):443–460. doi: 10.1016/s0022-2836(83)80344-1. [DOI] [PubMed] [Google Scholar]
  25. Tomschy A., Glockshuber R., Jaenicke R. Functional expression of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima in Escherichia coli. Authenticity and kinetic properties of the recombinant enzyme. Eur J Biochem. 1993 May 15;214(1):43–50. doi: 10.1111/j.1432-1033.1993.tb17894.x. [DOI] [PubMed] [Google Scholar]
  26. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  27. Vita C., Fontana A., Jaenicke R. Folding of thermolysin fragments. Hydrodynamic properties of isolated domains and subdomains. Eur J Biochem. 1989 Aug 15;183(3):513–518. doi: 10.1111/j.1432-1033.1989.tb21079.x. [DOI] [PubMed] [Google Scholar]
  28. Wetlaufer D. B. Folding of protein fragments. Adv Protein Chem. 1981;34:61–92. doi: 10.1016/s0065-3233(08)60518-5. [DOI] [PubMed] [Google Scholar]
  29. YPHANTIS D. A. EQUILIBRIUM ULTRACENTRIFUGATION OF DILUTE SOLUTIONS. Biochemistry. 1964 Mar;3:297–317. doi: 10.1021/bi00891a003. [DOI] [PubMed] [Google Scholar]
  30. Yutani K., Sato T., Ogasahara K., Miles E. W. Comparison of denaturation of tryptophan synthase alpha-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid. Arch Biochem Biophys. 1984 Mar;229(2):448–454. doi: 10.1016/0003-9861(84)90174-7. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES