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Preparation of heavy-atom derivatives using site-directed mutagenesis. Introduction of cysteine residues into γδ resolvase

Hatfull, GF and Sanderson, MR and Freemont, PS and Raccuia, PR and Grindley, NDF and Steitz, TA (1989) Preparation of heavy-atom derivatives using site-directed mutagenesis. Introduction of cysteine residues into γδ resolvase. Journal of Molecular Biology, 208 (4). 661 - 667. ISSN 0022-2836

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Abstract

The ability to determine protein structures by X-ray crystallography is often thwarted by the difficulty of finding isomorphous heavy-atom derivatives. The crystal structure of the site-specific recombinase, resolvase, has been difficult to determine for this reason. We have overcome this problem by introducing 13 single cysteine substitutions into the resolvase catalytic domain using oligonucleotide mutagenesis. The mutant proteins were screened for their ability to crystallize into the orthorhombic form and bind mercury ions isomorphously. Two mutant proteins provided excellent heavy-atom derivatives. This approach should be of general use and particularly helpful in cases where traditional methods have failed to produce a derivative. © 1989.

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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Hatfull, GFgfh@pitt.eduGFH
Sanderson, MR
Freemont, PS
Raccuia, PR
Grindley, NDF
Steitz, TA
Date: 20 August 1989
Date Type: Publication
Journal or Publication Title: Journal of Molecular Biology
Volume: 208
Number: 4
Page Range: 661 - 667
DOI or Unique Handle: 10.1016/0022-2836(89)90156-3
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Refereed: Yes
ISSN: 0022-2836
MeSH Headings: Amino Acid Sequence; Binding Sites; Crystallization; Cysteine; Ethylmercury Compounds; Molecular Sequence Data; Mutation; Nucleotidyltransferases; Oligonucleotides; Proteins; Transposases
PubMed ID: 2553982
Date Deposited: 23 Nov 2015 14:58
Last Modified: 02 Feb 2019 15:57
URI: http://d-scholarship.pitt.edu/id/eprint/26379

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