CHARACTERIZATION OF THE TRYPTOPHAN BINDING-SITE OF ESCHERICHIA-COLI TRYPTOPHAN HOLOREPRESSOR BY PHOSPHORESCENCE AND OPTICAL-DETECTION OF MAGNETIC-RESONANCE OF A TRYPTOPHAN-FREE MUTANT

被引:4
作者
LI, Z
MAKI, AH
EFTINK, MR
MAUN, CJ
MATTHEWS, CR
机构
[1] LAWRENCE LIVERMORE NATL LAB,DEPT CHEM,DAVIS,CA 95616
[2] UNIV MISSISSIPPI,DEPT CHEM,UNIVERSITY,MS 38677
[3] PENN STATE UNIV,DEPT CHEM,UNIVERSITY PK,PA 16802
关键词
D O I
10.1021/bi00039a048
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The L-tryptophan binding site of the Escherichia coli tryptophan holorepressor (trpR) is characterized by low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Measurements are made on a tryptophan-free mutant of trpR, W19/99F, in which both intrinsic tryptophan residues of apo-trpR have been replaced with phenylalanine. Thus, essentially all of the phosphorescence that is observed from trpR originates from the bound L-tryptophan corepressor. The phosphorescence and ODMR results for the bound corepressor agree quite well with those obtained previously for the corepressor site in both single tryptophan-containing mutants, W19F and W99F [Burns, L. E., & Maki, A. H. (1994) J. Fluorescence 4, 217-226]. A red shift of the L-tryptophan phosphorescence origin as well as a decrease in the D-E ODMR frequency result from an increase in the local polarizability upon binding at the corepressor binding site. A large decrease in the ODMR line widths signals a reduction of local heterogeneity upon binding. Subsequent binding of trpR to a self-complementary DNA sequence that mimics the trp operator, 5'-CGTACTAGTTAACTAGTACG-3', produces a further decrease in line widths and additional changes in the ODMR frequencies, attributable to an increase in both the D and E parameters. This result demonstrates that binding of holo-trpR to the operator affects the local environment of the bound corepressor.
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页码:12866 / 12870
页数:5
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