title: Structure and dynamics of staphylococcal nuclease mutants as studied by fluorescence quenching techniques.
abstract: Fluorescence techniques have been used to investigate the effect of mutations on the structure and dynamics of staphylococcal nuclease. An estimate of the accessibility to acrylamide of the enzyme's single tryptophan residue (Trp140) was obtained from the Stern-Volmer constant for fluorescence quenching. This was indicative of a partially buried tryptophan in the wild-type nuclease. Five single-site mutant nucleases (H124L, V66L, G88V, G79S and F76V) and one double mutant (V66L + G88V), with widely differing stabilities to denaturants, gave Stern-Volmer constants which were very similar to that of their parent enzyme. Studies of the temperature- and viscosity-dependence of quenching suggest that access by acrylamide to Trp140 is limited by diffusion rather than by protein structural fluctuations. Lifetime-resolved fluorescence anisotropy studies using steady-state instrumentation suggest that there is very little segmental motion of the Trp140; most of the anisotropy therefore decays due to protein rotation in the solution. Rotational correlation times for several nuclease mutants have been determined and these are very similar to that of the native nuclease. Thus it appears that these substitutions in the primary amino acid sequence, which have significant effects on the stability of the folded proteins, do not cause a significant change in the protein structure or dynamics around Trp140.
authors.affiliation: Biological Laboratory, University of Kent, Canterbury, UK.
authors.forename: G
authors.initials: G
authors.lastname: Wright
authors.forename: R B
authors.initials: RB
authors.lastname: Freedman
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000302:isolation & purification; D015394:Molecular Structure; D009154:Mutation; D013696:Temperature; D013816:Thermodynamics; D014783:Viscosity
chemical_list: D008836:Micrococcal Nuclease