title: Preparation of heavy-atom derivatives using site-directed mutagenesis. Introduction of cysteine residues into gamma delta resolvase.
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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, New Haven, CT 06511.
authors.forename: G F
authors.initials: GF
authors.lastname: Hatfull
authors.forename: M R
authors.initials: MR
authors.lastname: Sanderson
authors.forename: P S
authors.initials: PS
authors.lastname: Freemont
authors.forename: P R
authors.initials: PR
authors.lastname: Raccuia
authors.forename: N D
authors.initials: ND
authors.lastname: Grindley
authors.forename: T A
authors.initials: TA
authors.lastname: Steitz
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003460:Crystallization; D003545:Cysteine; D005035:Ethylmercury Compounds; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases; D009841:Oligonucleotides; D011506:Proteins; D019895:Transposases
chemical_list: D005035:Ethylmercury Compounds; D009841:Oligonucleotides; D011506:Proteins; D009713:Nucleotidyltransferases; D019895:Transposases; D003545:Cysteine