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. 1989 Dec;86(24):9966–9970. doi: 10.1073/pnas.86.24.9966

An experimental approach to testing modular evolution: directed replacement of alpha-helices in a bacterial protein.

R F DuBose 1, D L Hartl 1
PMCID: PMC298623  PMID: 2690081

Abstract

We have used oligonucleotide site-directed mutagenesis to ask whether certain structural motifs in proteins are determined mainly by local interactions among amino acids. Multiple consecutive amino acids in three alpha-helices in the alkaline phosphatase (EC 3.1.3.1) of Escherichia coli have been replaced with helical sequences from four other sources. Altogether, 12 distinct helical replacements were created, 9 of which retain enzymatic activity. Most short stretches of helical sequence can be replaced with unrelated helical sequences without eliminating enzyme activity. Replacements of the carboxyl half of an alpha-helix are less harmful than those of the amino half, and the two together are synergistic rather than additive. These results are consistent with the hypothesis that proteins originally evolved by the assembly of small functional folding units.

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Selected References

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

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Brenner S. The molecular evolution of genes and proteins: a tale of two serines. Nature. 1988 Aug 11;334(6182):528–530. doi: 10.1038/334528a0. [DOI] [PubMed] [Google Scholar]
  3. Butler-Ransohoff J. E., Kendall D. A., Kaiser E. T. Use of site-directed mutagenesis to elucidate the role of arginine-166 in the catalytic mechanism of alkaline phosphatase. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4276–4278. doi: 10.1073/pnas.85.12.4276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chaidaroglou A., Brezinski D. J., Middleton S. A., Kantrowitz E. R. Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase. Biochemistry. 1988 Nov 1;27(22):8338–8343. doi: 10.1021/bi00422a008. [DOI] [PubMed] [Google Scholar]
  5. Darnell J. E., Doolittle W. F. Speculations on the early course of evolution. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1271–1275. doi: 10.1073/pnas.83.5.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DuBose R. F., Dykhuizen D. E., Hartl D. L. Genetic exchange among natural isolates of bacteria: recombination within the phoA gene of Escherichia coli. Proc Natl Acad Sci U S A. 1988 Sep;85(18):7036–7040. doi: 10.1073/pnas.85.18.7036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fitch W. M. The molecular evolution of cytochrome c in eukaryotes. J Mol Evol. 1976 Jun 23;8(1):13–40. doi: 10.1007/BF01738880. [DOI] [PubMed] [Google Scholar]
  8. Gilbert W. Genes-in-pieces revisited. Science. 1985 May 17;228(4701):823–824. doi: 10.1126/science.4001923. [DOI] [PubMed] [Google Scholar]
  9. Gilbert W. Why genes in pieces? Nature. 1978 Feb 9;271(5645):501–501. doi: 10.1038/271501a0. [DOI] [PubMed] [Google Scholar]
  10. Giniger E., Ptashne M. Transcription in yeast activated by a putative amphipathic alpha helix linked to a DNA binding unit. Nature. 1987 Dec 17;330(6149):670–672. doi: 10.1038/330670a0. [DOI] [PubMed] [Google Scholar]
  11. Houghton J. E., O'Donovan G. A., Wild J. R. Reconstruction of an enzyme by domain substitution effectively switches substrate specificity. Nature. 1989 Mar 9;338(6211):172–174. doi: 10.1038/338172a0. [DOI] [PubMed] [Google Scholar]
  12. Hynes T. R., Kautz R. A., Goodman M. A., Gill J. F., Fox R. O. Transfer of a beta-turn structure to a new protein context. Nature. 1989 May 4;339(6219):73–76. doi: 10.1038/339073a0. [DOI] [PubMed] [Google Scholar]
  13. Jung A., Sippel A. E., Grez M., Schütz G. Exons encode functional and structural units of chicken lysozyme. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5759–5763. doi: 10.1073/pnas.77.10.5759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kunisawa T., Horimoto K., Otsuka J. Accumulation pattern of amino acid substitutions in protein evolution. J Mol Evol. 1987;24(4):357–365. doi: 10.1007/BF02134134. [DOI] [PubMed] [Google Scholar]
  15. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lazzaroni J. C., Atlan D., Portalier R. C. Excretion of alkaline phosphatase by Escherichia coli K-12 pho constitutive mutants transformed with plasmids carrying the alkaline phosphatase structural gene. J Bacteriol. 1985 Dec;164(3):1376–1380. doi: 10.1128/jb.164.3.1376-1380.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matthews B. W., Remington S. J. The three dimensional structure of the lysozyme from bacteriophage T4. Proc Natl Acad Sci U S A. 1974 Oct;71(10):4178–4182. doi: 10.1073/pnas.71.10.4178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ohno S. Early genes that were oligomeric repeats generated a number of divergent domains on their own. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6486–6490. doi: 10.1073/pnas.84.18.6486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ohno S. Evolution from primordial oligomeric repeats to modern coding sequences. J Mol Evol. 1987;25(4):325–329. doi: 10.1007/BF02603117. [DOI] [PubMed] [Google Scholar]
  20. Regan L., DeGrado W. F. Characterization of a helical protein designed from first principles. Science. 1988 Aug 19;241(4868):976–978. doi: 10.1126/science.3043666. [DOI] [PubMed] [Google Scholar]
  21. Sowadski J. M., Handschumacher M. D., Murthy H. M., Foster B. A., Wyckoff H. W. Refined structure of alkaline phosphatase from Escherichia coli at 2.8 A resolution. J Mol Biol. 1985 Nov 20;186(2):417–433. doi: 10.1016/0022-2836(85)90115-9. [DOI] [PubMed] [Google Scholar]
  22. Südhof T. C., Russell D. W., Goldstein J. L., Brown M. S., Sanchez-Pescador R., Bell G. I. Cassette of eight exons shared by genes for LDL receptor and EGF precursor. Science. 1985 May 17;228(4701):893–895. doi: 10.1126/science.3873704. [DOI] [PubMed] [Google Scholar]
  23. Traut T. W. Do exons code for structural or functional units in proteins? Proc Natl Acad Sci U S A. 1988 May;85(9):2944–2948. doi: 10.1073/pnas.85.9.2944. [DOI] [PMC free article] [PubMed] [Google Scholar]

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