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
. 1988 Sep;85(18):6607–6611. doi: 10.1073/pnas.85.18.6607

In vitro assay for protein-protein interaction: carboxyl-terminal 40 residues of simian virus 40 structural protein VP3 contain a determinant for interaction with VP1.

E Gharakhanian 1, J Takahashi 1, J Clever 1, H Kasamatsu 1
PMCID: PMC282026  PMID: 2842781

Abstract

Intermolecular interactions between polypeptide chains play essential roles in the functioning of proteins. We describe here an in vitro assay system for identifying and characterizing such interactions. Such interactions are difficult to study in vivo. We have translated synthetic, nonmethyl-capped RNAs in a cell-free protein-synthesizing system. The translation products were allowed to interact posttranslationally to form protein-protein complexes. The chemical nature of the protein interaction(s) was determined by coimmunoprecipitation of associating proteins, sedimentation through sucrose gradients, followed by NaDodSO4/polyacrylamide gel electrophoresis or by nonreducing NaDodSO4/polyacrylamide gel electrophoresis. The system has been utilized to show the self-assembly of monomeric VP1, the major structural protein of simian virus 40, into disulfide-linked pentamers and to show the noncovalent interaction of another structural protein, VP3, with VP1 at low monomer concentrations. Additionally, we show that the carboxyl-terminal 40 amino acids of VP3 are essential and sufficient for its interaction with VP1 in vitro. The in vitro assay system described here provides a method for identifying the domains involved in, and the molecular nature of, protein-protein interactions, which play an important role in such biological phenomena as replication, transcription, translation, transport, ligand binding, and assembly.

Full text

PDF
6607

Images in this article

6608Image
on p.6608
6608Image
on p.6608
6609Image
on p.6609
6609Image
on p.6609
6609Image
on p.6609
6610Image
on p.6610
6610Image
on p.6610
6610Image
on p.6610
6610Image
on p.6610

Selected References

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

  1. Anfinsen C. B. Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223–230. doi: 10.1126/science.181.4096.223. [DOI] [PubMed] [Google Scholar]
  2. Blasquez V., Stein A., Ambrose C., Bina M. Simian virus 40 protein VP1 is involved in spacing nucleosomes in minichromosomes. J Mol Biol. 1986 Sep 5;191(1):97–106. doi: 10.1016/0022-2836(86)90425-0. [DOI] [PubMed] [Google Scholar]
  3. Brady J. N., Consigli R. A. Chromatographic separation of the polyoma virus proteins and renaturation of the isolated VP1 major capsid protein. J Virol. 1978 Aug;27(2):436–442. doi: 10.1128/jvi.27.2.436-442.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brady J. N., Lavialle C., Salzman N. P. Efficient transcription of a compact nucleoprotein complex isolated from purified simian virus 40 virions. J Virol. 1980 Aug;35(2):371–381. doi: 10.1128/jvi.35.2.371-381.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fernandez-Munoz R., Coca-Prados M., Hsu M. T. Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells. J Virol. 1979 Feb;29(2):612–623. doi: 10.1128/jvi.29.2.612-623.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fowler A. V., Zabin I. Purification, structure, and properties of hybrid beta-galactosidase proteins. J Biol Chem. 1983 Dec 10;258(23):14354–14358. [PubMed] [Google Scholar]
  7. Gharakhanian E., Takahashi J., Kasamatsu H. The carboxyl 35 amino acids of SV40 Vp3 are essential for its nuclear accumulation. Virology. 1987 Apr;157(2):440–448. doi: 10.1016/0042-6822(87)90286-8. [DOI] [PubMed] [Google Scholar]
  8. Hunter T. Functional characterization of the early and late mRNAs of simian virus 40. Virology. 1979 Jun;95(2):511–522. doi: 10.1016/0042-6822(79)90505-1. [DOI] [PubMed] [Google Scholar]
  9. Kasamatsu H., Nehorayan A. Vp1 affects intracellular localization of Vp3 polypeptide during simian virus 40 infection. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2808–2812. doi: 10.1073/pnas.76.6.2808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kim P. S., Baldwin R. L. Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Annu Rev Biochem. 1982;51:459–489. doi: 10.1146/annurev.bi.51.070182.002331. [DOI] [PubMed] [Google Scholar]
  11. Kosower N. S., Kosower E. M. The glutathione status of cells. Int Rev Cytol. 1978;54:109–160. doi: 10.1016/s0074-7696(08)60166-7. [DOI] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. La Bella F., Vesco C. Late modifications of simian virus 40 chromatin during the lytic cycle occur in an immature form of virion. J Virol. 1980 Mar;33(3):1138–1150. doi: 10.1128/jvi.33.3.1138-1150.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  16. Lin W., Hata T., Kasamatsu H. Subcellular distribution of viral structural proteins during simian virus 40 infection. J Virol. 1984 May;50(2):363–371. doi: 10.1128/jvi.50.2.363-371.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lin W., Shurgot J. L., Kasamatsu H. The synthesis and transport of SV40 structural proteins. Virology. 1986 Oct 15;154(1):108–120. doi: 10.1016/0042-6822(86)90434-4. [DOI] [PubMed] [Google Scholar]
  18. Llopis R., Stark G. R. Two deletions within genes for simian virus 40 structural proteins VP2 and VP3 lead to formation of abnormal transcriptional complexes. J Virol. 1981 Apr;38(1):91–103. doi: 10.1128/jvi.38.1.91-103.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moyne G., Harper F., Saragosti S., Yaniv M. Absence of nucleosomes in a histone-containing nucleoprotein complex obtained by dissociation of purified SV40 virions. Cell. 1982 Aug;30(1):123–130. doi: 10.1016/0092-8674(82)90018-6. [DOI] [PubMed] [Google Scholar]
  21. Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
  22. Ozer H. L., Tegtmeyer P. Synthesis and assembly of simian virus 40. II. Synthesis of the major capsid protein and its incorporation into viral particles. J Virol. 1972 Jan;9(1):52–60. doi: 10.1128/jvi.9.1.52-60.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rüther U., Müller-Hill B. Easy identification of cDNA clones. EMBO J. 1983;2(10):1791–1794. doi: 10.1002/j.1460-2075.1983.tb01659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Salunke D. M., Caspar D. L., Garcea R. L. Self-assembly of purified polyomavirus capsid protein VP1. Cell. 1986 Sep 12;46(6):895–904. doi: 10.1016/0092-8674(86)90071-1. [DOI] [PubMed] [Google Scholar]
  25. Wychowski C., Benichou D., Girard M. The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence. J Virol. 1987 Dec;61(12):3862–3869. doi: 10.1128/jvi.61.12.3862-3869.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yu M. H., King J. Single amino acid substitutions influencing the folding pathway of the phage P22 tail spike endorhamnosidase. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6584–6588. doi: 10.1073/pnas.81.21.6584. [DOI] [PMC free article] [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