Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Aug 1;90(15):7010–7014. doi: 10.1073/pnas.90.15.7010

Genetic fusion of subunits of a dimeric protein substantially enhances its stability and rate of folding.

H Liang 1, W S Sandberg 1, T C Terwilliger 1
PMCID: PMC47065  PMID: 8346210

Abstract

The gene V protein of bacteriophage f1 is a single-stranded DNA and RNA-binding protein composed of two identical subunits. We have constructed single-chain variants of the protein using short peptide linkers of five or six amino acids to connect the carboxyl terminus of one monomer to the amino terminus of the second monomer. The resulting subunit-fusion gene V proteins were found to bind single-stranded DNA nearly as tightly as the wild-type protein. Denaturation measurements show that the subunit-fusion gene V proteins are 5 kcal/mol (1 kcal = 4.18 kJ) more stable than the wild-type protein at a protein concentration of 10 microM. The rate of unfolding of the protein is essentially unaffected by the fusion of monomeric subunits, whereas the rate of folding is greatly enhanced. Our results suggest a simple way of obtaining a substantial thermodynamic stabilization for some oligomeric proteins.

Full text

PDF
7010

Images in this article

7011Fig. 1
on p.7011

Selected References

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

  1. Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990 Feb 20;211(4):943–958. doi: 10.1016/0022-2836(90)90085-Z. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bizub D., Weber I. T., Cameron C. E., Leis J. P., Skalka A. M. A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains. J Biol Chem. 1991 Mar 15;266(8):4951–4958. [PubMed] [Google Scholar]
  4. Brayer G. D., McPherson A. Refined structure of the gene 5 DNA binding protein from bacteriophage fd. J Mol Biol. 1983 Sep 15;169(2):565–596. doi: 10.1016/s0022-2836(83)80065-5. [DOI] [PubMed] [Google Scholar]
  5. Corbett R. J., Roche R. S. Use of high-speed size-exclusion chromatography for the study of protein folding and stability. Biochemistry. 1984 Apr 10;23(8):1888–1894. doi: 10.1021/bi00303a047. [DOI] [PubMed] [Google Scholar]
  6. Davis N. G., Boeke J. D., Model P. Fine structure of a membrane anchor domain. J Mol Biol. 1985 Jan 5;181(1):111–121. doi: 10.1016/0022-2836(85)90329-8. [DOI] [PubMed] [Google Scholar]
  7. Davis N. G., Model P. An artificial anchor domain: hydrophobicity suffices to stop transfer. Cell. 1985 Jun;41(2):607–614. doi: 10.1016/s0092-8674(85)80033-7. [DOI] [PubMed] [Google Scholar]
  8. Day L. A. Circular dichroism and ultraviolet absorption of a deoxyribonucleic acid binding protein of filamentous bacteriophage. Biochemistry. 1973 Dec 18;12(26):5329–5339. doi: 10.1021/bi00750a017. [DOI] [PubMed] [Google Scholar]
  9. Folkers P. J., van Duynhoven J. P., Jonker A. J., Harmsen B. J., Konings R. N., Hilbers C. W. Sequence-specific 1H-NMR assignment and secondary structure of the Tyr41----His mutant of the single-stranded DNA binding protein, gene V protein, encoded by the filamentous bacteriophage M13. Eur J Biochem. 1991 Dec 5;202(2):349–360. doi: 10.1111/j.1432-1033.1991.tb16382.x. [DOI] [PubMed] [Google Scholar]
  10. Gittelman M. S., Matthews C. R. Folding and stability of trp aporepressor from Escherichia coli. Biochemistry. 1990 Jul 31;29(30):7011–7020. doi: 10.1021/bi00482a009. [DOI] [PubMed] [Google Scholar]
  11. Glockshuber R., Malia M., Pfitzinger I., Plückthun A. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry. 1990 Feb 13;29(6):1362–1367. doi: 10.1021/bi00458a002. [DOI] [PubMed] [Google Scholar]
  12. Hallewell R. A., Laria I., Tabrizi A., Carlin G., Getzoff E. D., Tainer J. A., Cousens L. S., Mullenbach G. T. Genetically engineered polymers of human CuZn superoxide dismutase. Biochemistry and serum half-lives. J Biol Chem. 1989 Mar 25;264(9):5260–5268. [PubMed] [Google Scholar]
  13. Ito K., Date T., Wickner W. Synthesis, assembly into the cytoplasmic membrane, and proteolytic processing of the precursor of coliphage M13 coat protein. J Biol Chem. 1980 Mar 10;255(5):2123–2130. [PubMed] [Google Scholar]
  14. Jaenicke R. Folding and association of proteins. Prog Biophys Mol Biol. 1987;49(2-3):117–237. doi: 10.1016/0079-6107(87)90011-3. [DOI] [PubMed] [Google Scholar]
  15. Liang H., Terwilliger T. C. Reversible denaturation of the gene V protein of bacteriophage f1. Biochemistry. 1991 Mar 19;30(11):2772–2782. doi: 10.1021/bi00225a006. [DOI] [PubMed] [Google Scholar]
  16. Maloy S. R., Nunn W. D. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. doi: 10.1128/jb.145.2.1110-1111.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matsumura M., Becktel W. J., Levitt M., Matthews B. W. Stabilization of phage T4 lysozyme by engineered disulfide bonds. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6562–6566. doi: 10.1073/pnas.86.17.6562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McLachlan A. D. Gene duplication and the origin of repetitive protein structures. Cold Spring Harb Symp Quant Biol. 1987;52:411–420. doi: 10.1101/sqb.1987.052.01.048. [DOI] [PubMed] [Google Scholar]
  19. McLachlan A. D. Repeating sequences and gene duplication in proteins. J Mol Biol. 1972 Mar 14;64(2):417–437. doi: 10.1016/0022-2836(72)90508-6. [DOI] [PubMed] [Google Scholar]
  20. Nishikawa S., Adiwinata J., Morioka H., Fujimura T., Tanaka T., Uesugi S., Hakoshima T., Tomita K., Nakagawa S., Ikehara M. A thermoresistant mutant of ribonuclease T1 having three disulfide bonds. Protein Eng. 1990 Apr;3(5):443–448. doi: 10.1093/protein/3.5.443. [DOI] [PubMed] [Google Scholar]
  21. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  22. Page M. I., Jencks W. P. Entropic contributions to rate accelerations in enzymic and intramolecular reactions and the chelate effect. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1678–1683. doi: 10.1073/pnas.68.8.1678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pantoliano M. W., Bird R. E., Johnson S., Asel E. D., Dodd S. W., Wood J. F., Hardman K. D. Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli. Biochemistry. 1991 Oct 22;30(42):10117–10125. doi: 10.1021/bi00106a007. [DOI] [PubMed] [Google Scholar]
  24. Sandberg W. S., Terwilliger T. C. Energetics of repacking a protein interior. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1706–1710. doi: 10.1073/pnas.88.5.1706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sandberg W. S., Terwilliger T. C. Influence of interior packing and hydrophobicity on the stability of a protein. Science. 1989 Jul 7;245(4913):54–57. doi: 10.1126/science.2787053. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tang J., James M. N., Hsu I. N., Jenkins J. A., Blundell T. L. Structural evidence for gene duplication in the evolution of the acid proteases. Nature. 1978 Feb 16;271(5646):618–621. doi: 10.1038/271618a0. [DOI] [PubMed] [Google Scholar]
  28. Tasayco M. L., Carey J. Ordered self-assembly of polypeptide fragments to form nativelike dimeric trp repressor. Science. 1992 Jan 31;255(5044):594–597. doi: 10.1126/science.1736361. [DOI] [PubMed] [Google Scholar]
  29. Terwilliger T. C. Construction of a synthetic variant of the bacteriophage f1 gene V by assembling oligodeoxynucleotides corresponding to only one strand of DNA. Gene. 1988 Nov 15;71(1):41–47. doi: 10.1016/0378-1119(88)90075-3. [DOI] [PubMed] [Google Scholar]
  30. Terwilliger T. C., Fulford W. D., Zabin H. B. A genetic selection for temperature-sensitive variants of the gene V protein of bacteriophage f1. Nucleic Acids Res. 1988 Sep 26;16(18):9027–9039. doi: 10.1093/nar/16.18.9027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Toth M. J., Schimmel P. Internal structural features of E. coli glycyl-tRNA synthetase examined by subunit polypeptide chain fusions. J Biol Chem. 1986 May 25;261(15):6643–6646. [PubMed] [Google Scholar]
  33. Weeds A. G., McLachlan A. D. Structural homology of myosin alkali light chains, troponin C and carp calcium binding protein. Nature. 1974 Dec 20;252(5485):646–649. doi: 10.1038/252646a0. [DOI] [PubMed] [Google Scholar]
  34. Zabin H. B., Terwilliger T. C. Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein. J Mol Biol. 1991 May 20;219(2):257–275. doi: 10.1016/0022-2836(91)90566-o. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES