Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids.


Abstract

We report steady-state and time-resolved fluorescence studies with the single tryptophan protein, Staphylococcus aureus A, and several of its site-directed mutants. A couple of these mutants, nuclease-conA and nuclease-conA-S28G (which are hybrid proteins containing a six amino acid beta-turn substitute from concanavalin A), are found to have a much lower thermodynamic stability than the wild type. The thermal transition temperatures for nuclease-conA and S28G are 32.8 and 30.5 degrees C, which are about 20 degrees C lower than the Tm for wild-type nuclease A. These mutant proteins also are denatured by a much lower concentration of the denaturants urea and guanidine hydrochloride. We also show that an unfolding transition in the structure of the nuclease-conA hybrids can be induced by relatively low hydrostatic pressure (approximately 700 bar). The free energy for unfolding of nuclease-conA (and nuclease-conA-S28G) is found to be only 1.4 kcal/mol (and 1.2 kcal/mol) by thermal, urea, guanidine hydrochloride, and pressure unfolding. Time-resolved fluorescence intensity and anisotropy measurements with nuclease-conA-S28G show the temperature-, urea-, and pressure-perturbed states each to have a reduced average intensity decay time and to depolarize with a rotational correlation time of approximately 1.0 ns (as compared to a rotational correlation time of 11 ns for the native form of nuclease-conA-S28G at 20 degrees C). Study holds ProTherm entries: 378, 379, 380, 381, 382 Extra Details: Staphylococcus nuclease; conformational stability; free energy

Submission Details

ID: rayqfKrv3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Eftink MR;Ghiron CA;Kautz RA;Fox RO,Biochemistry (1991) Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids. PMID:1991099
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