title: Effects of temperature on the fluorescence intensity and anisotropy decays of staphylococcal nuclease and the less stable nuclease-conA-SG28 mutant.
abstract: Frequency-domain fluorescence spectroscopy was used to investigate the effects of temperature on the intensity and anisotropy decays of the single tryptophan residues of Staphylococcal nuclease A and its nuclease-conA-SG28 mutant. This mutant has the beta-turn forming hexapeptide, Ser-Gly-Asn-Gly-Ser-Pro, substituted for the pentapeptide Tyr-Lys-Gly-Gln-Pro at positions 27-31. The intensity decays were analyzed in terms of a sum of exponentials and with Lorentzian distributions of decay times. The anisotropy decays were analyzed in terms of a sum of exponentials. Both the intensity and anisotropy decay parameters strongly depend on temperature near the thermal transitions of the proteins. Significant differences in the temperature stability of Staphylococcal nuclease and the mutant exist; these proteins show characteristic thermal transition temperatures (Tm) of 51 and 30 degrees C, respectively, at pH 7. The temperature dependence of the intensity decay data are shown to be consistent with a two-state unfolding model. For both proteins, the longer rotational correlation time, due to overall rotational diffusion, decreases dramatically at the transition temperature, and the amplitude of the shorter correlation time increases, indicating increased segmental motions of the single tryptophan residue. The mutant protein appears to have a slightly larger overall rotational correlation time and to show slightly more segmental motion of its Trp than is the case for the wild-type protein.
authors.affiliation: Department of Chemistry, University of Mississippi, University 38677.
authors.forename: M R
authors.initials: MR
authors.lastname: Eftink
authors.forename: I
authors.initials: I
authors.lastname: Gryczynski
authors.forename: W
authors.initials: W
authors.lastname: Wiczk
authors.forename: G
authors.initials: G
authors.lastname: Laczko
authors.forename: J R
authors.initials: JR
authors.lastname: Lakowicz
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005454:Fluorescence Polarization; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics
chemical_list: D008836:Micrococcal Nuclease