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Proceedings Paper

Fluorescence and conformational stability studies of S. nuclease A and its site-directed mutants
Author(s): Maurice R. Eftink; Camillo A. Ghiron; Ignacy Gryczynski; Wieslaw M. Wiczk; Gabor Laczko; Joseph R. Lakowicz
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Paper Abstract

We report fluorescence studies with the single trp protein, S. nucelase A, and several of its site-directed mutants. One of these mutants, PA56, which has an alanine at position 56 in place of proline, has a much lower structural stability than the wild type. This is demonstrated by the much lower Tm (30 degrees C) for PA56 than for the wild type (52 degrees C) and by a much lower (urea)1/2 for denaturation of the mutant. Also we show that PA56 can be unfolded by relatively low hydrostatic pressure (~700 bar). The free energy for unfolding of PA56 is found to be only 1.3 kcal/mole (at 20 degrees C) by thermal, urea, quanidine and pressure unfolding. Fluorescence lifetime measurements with wild type nuclease and several of its mutants show non-exponential decay kinetics. The fluorescence decay profiles are similar for the native state of each protein and the decay data at various temperatures generally reveal differences in the Tm for the various mutants. Anisotropy decay data are analyzed in terms of two rotational correlation times, a longer one for overall rotation of the protein and a shorter one for rapid, segemental motion of the trp residue. The mutant PA56 can be easily denatured by temperature, pressure or urea, and anisotropy decay data for these various denatured forms are reported.

Paper Details

Date Published: 1 May 1990
PDF: 7 pages
Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); doi: 10.1117/12.17695
Show Author Affiliations
Maurice R. Eftink, Univ. of Mississippi (United States)
Camillo A. Ghiron, Univ. of Missouri/Columbia (United States)
Ignacy Gryczynski, Univ. of Maryland (United States)
Wieslaw M. Wiczk, Univ. of Maryland (United States)
Gabor Laczko, Univ. of Maryland (United States)
Joseph R. Lakowicz, Univ. of Maryland (United States)

Published in SPIE Proceedings Vol. 1204:
Time-Resolved Laser Spectroscopy in Biochemistry II
Joseph R. Lakowicz, Editor(s)

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