Effects of Temperature on the Fluorescence Intensity and Anisotropy Decays of Staphylococcal Nuclease and the Less Stable Nuclease-ConA-SG28 Mutant

Maurice R. Eftink, Ignacy Gryczynski, Wieslaw Wiczk, Gabor Laczko, Joseph R. Lakowicz

Research output: Contribution to journalArticle

37 Scopus citations

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 /3-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 °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.

Original languageEnglish
Pages (from-to)8945-8953
Number of pages9
JournalBiochemistry
Volume30
Issue number37
DOIs
StatePublished - 1 Sep 1991

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