Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids.
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).
Eftink
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MR
Department of Chemistry, University of Mississippi, University 38677.
Ghiron
C A
CA
Kautz
R A
RA
Fox
R O
RO
D000178:Acrylamides; D003208:Concanavalin A; D004252:DNA Mutational Analysis; D006874:Hydrostatic Pressure; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000648:ultrastructure*; D011487:Protein Conformation; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000201:enzymology; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics
D000178:Acrylamides; D012997:Solvents; D003208:Concanavalin A; D008836:Micrococcal Nuclease