title: The impact of single cysteine residue mutations on the replication terminator protein.
abstract: We report the structural and biophysical consequences of cysteine substitutions in the DNA-binding replication terminator protein (RTP) of Bacillus subtilis, that resulted in an optimised RTP mutant suitable for structural studies. The cysteine residue 110 was replaced with alanine, valine or serine. Protein secondary structure and stability (using circular dichroism spectropolarimetry), self-association (using analytical ultracentrifugation), and DNA-binding measurements revealed RTP.C110S to be the most similar mutant to wild-type RTP. The C110A and C110V.RTP mutants were less soluble, less stable and showed lower DNA-binding affinity. The structure of RTP.C110S, solved to 2.5A resolution using crystallographic methods, showed no major structural perturbation due to the mutation. Heteronuclear NMR spectroscopic studies revealed subtle differences in the electronic environment about the site of mutation. The study demonstrates the suitability of serine as a substitute for cysteine in RTP and the high sensitivity of protein behaviour to single amino acid substitutions.
authors.affiliation: Department of Pharmacology, Western Australian Institute for Medical Research, University of Western Australia, Perth, WA 6009, Australia.
authors.forename: J P
authors.initials: JP
authors.lastname: Vivian
authors.forename: A F
authors.initials: AF
authors.lastname: Hastings
authors.forename: I G
authors.initials: IG
authors.lastname: Duggin
authors.forename: R G
authors.initials: RG
authors.lastname: Wake
authors.forename: M C J
authors.initials: MC
authors.lastname: Wilce
authors.forename: J A
authors.initials: JA
authors.lastname: Wilce
mesh_terms: D001426:Bacterial Proteins; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C063112:rtP protein, Bacillus subtilis; D003545:Cysteine