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Directed Mutagenesis of Dihydrofolate Reductase

Villafranca, Jesus E. and Howell, Elizabeth E. and Voet, Donald H. and Strobel, Marjorie C. and Ogden, Richard C. and Abelson, John N. and Kraut, Joseph (1983) Directed Mutagenesis of Dihydrofolate Reductase. Science, 222 (4625). pp. 782-788. ISSN 0036-8075. http://resolver.caltech.edu/CaltechAUTHORS:20150205-094225275

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Abstract

Three mutations of the enzyme dihydrofolate reductase were constructed by oligonucleotide-directed mutagenesis of the cloned Escherichia coli gene. The mutations--at residue 27, aspartic acid replaced with asparagine; at residue 39, proline replaced with cysteine; and at residue 95, glycine replaced with alanine--were designed to answer questions about the relations between molecular structure and function that were raised by the x-ray crystal structures. Properties of the mutant proteins show that Asp-27 is important for catalysis and that perturbation of the local structure at a conserved cis peptide bond following Gly-95 abolishes activity. Substitution of cysteine for proline at residue 39 results in the appearance of new forms of the enzyme that correspond to various oxidation states of the cysteine. One of these forms probably represents a species cross-linked by an intrachain disulfide bridge between the cysteine at position 85 and the new cysteine at position 39.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1126/science.6356360DOIArticle
http://www.sciencemag.org/content/222/4625/782.abstractPublisherArticle
http://www.jstor.org/stable/1691146JSTORArticle
Additional Information:© 1983 American Association for the Advancement of Science. We thank R. Matthews for providing the E. coli strain CV634, which contained the plasmid CV634; P. Price for the cysteic acid determinations; and R. Aust, E. Dyke, F. Lopez, and M. E. Ford for technical assistance. Supported in part by contract N00014-88-C-1482 (to J.K.) from the Office of Naval Research, Public Health Service fellowship F32 GM09375 (to E.E.H.), and NIH grants GM10928 and CA17374 (to J.K.).
Funders:
Funding AgencyGrant Number
Office of Naval Research (ONR)N00014-88-C-1482
U.S. Public Health ServiceF32 GM09375
NIHGM10928
NIHCA17374
Record Number:CaltechAUTHORS:20150205-094225275
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20150205-094225275
Official Citation:Directed mutagenesis of dihydrofolate reductase JE Villafranca, EE Howell, DH Voet, MS Strobel, RC Ogden, JN Abelson, and J Kraut Science 18 November 1983: 222 (4625), 782-788. [DOI:10.1126/science.6356360]
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:54412
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:05 Feb 2015 23:04
Last Modified:05 Feb 2015 23:04

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