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Title:Characterization of effect of tryptophan ontrp repressor subunit association using fluorescence spectroscopy
Author(s):Fernando, Teresa M.
Doctoral Committee Chair(s):Royer, C.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Abstract:trp repressor is an allosteric protein which regulates the expression of the trp operon in E. coli. Binding of tryptophan to trp repressor causes it to bind with high affinity to trp operator DNA. The active form of the protein is accepted to be dimeric. Preliminary results from our lab and others suggested that trp repressor exists as tetramer and higher order oligomeric forms, as well as the active dimer. Thus, the main objective of this research was to characterize the effect of tryptophan on the association of trp repressor subunits in order to determine whether the equilibria between higher order oligomeric forms and dimer may be involved in the allosteric mechanism of trp repressor. The extent of trp repressor subunit association was examined using fluorescence anisotropy of trp repressor labelled with the fluorescent dye, dansyl. Steady state and time resolved methods were used. The results obtained suggest that trp repressor exists as a mixture higher order oligomers and dimer at micromolar concentrations of protein. Tryptophan binding induces a time dependent dissociation to dimer. The buffer conditions affect the time it takes for dissociation. Denaturation curves were generated by measuring anisotropy as a function of urea concentration. One transition from tetramer to unfolded monomer was observed in the absence of tryptophan. Two transitions were observed in the presence of tryptophan: Tetramer to dimer at low urea concentrations; and dimer to monomer at high urea concentrations. A simulation of the fraction of total protein which is tryptophan bound dimer as a function of tryptophan concentration was performed using a model which incorporated tetrameric trp repressor and the free energies obtained from the denaturation curves. The fraction of the active dimer rose more sharply with increasing tryptophan concentration than when tetramer was not included in the model. To summarize, the results from this research show that trp repressor exists as a mixture of higher oligomeric forms and dimer, and a possible role for this mixture is to make the regulation of expression of the trp operon more sensitive to changes in the level of tryptophan.
Issue Date:1991
Rights Information:Copyright 1991 Fernando, Teresa M.
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9210800
OCLC Identifier:(UMI)AAI9210800

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