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dc.contributor.advisor Matthews, Kathleen S.
dc.creatorChou, Wei-Yuan
dc.date.accessioned 2009-06-04T00:24:43Z
dc.date.available 2009-06-04T00:24:43Z
dc.date.issued 1988
dc.identifier.urihttps://hdl.handle.net/1911/16331
dc.description.abstract The interactions of Escherichia coli trp aporepressor and its ligands, tryptophan and trp operator DNA, were examined. To avoid the interference of fluorescence from the ligand, a hydrophobicity-dependent fluorophore, 8-anilino-1-naphthalenesulfonate (ANS), was used to monitor tryptophan binding. The fluorescence decrease of ANS caused by tryptophan competitive displacement was utilized to measure the affinity of tryptophan and its aporepressor. The results analyzed by the method of Horovitz and Levitzki (1987) showed the equilibrium dissociation constant to be 3 $\times$ 10$\sp{-5}$ M. This value is similar to the results obtained from equilibrium dialysis and other spectrometric methods. The kinetic parameters were measured by monitoring the tryptophan fluorescence difference directly on stopped-flow fluorospectrometer. The dissociation rate constant of tryptophan from trp aporepressor is about 50 s$\sp{-1}$. The operator binding assay was performed by gel retardation electrophoresis developed by Carey (1987). The apparent equilibrium dissociation constants for trp repressor and 40 or 90 base pair (bp) operator-containing DNA are identical. A dissociation rate of 5 $\times$ 10$\sp{-2}$ s$\sp{-1}$ was obtained by the displacement experiment with unlabeled operator DNA. This result was consistent with the value from nitrocellulose filter binding assay (Klig et al., 1987). Three point mutations of trp aporepressor, each with a serine to cysteine at positions 67, 86 and 88, were constructed by oligonucleotide-directed site-specific mutagenesis. Taking the advantage of the similarities between hydroxyl and sulfhydryl groups, these substitutions were made to provide sites to introduce fluorescent probes into the mutant proteins. However, both tryptophan and operator DNA binding affinities for all three mutants were decreased. These results may reflect the complexity within this region which includes the amino acid residues in the tryptophan binding site, DNA recognition site and hydrophobic brace (Zhang et al., 1987). Results from ultracentrifugation, polarization of fluorescence and gel filtration indicate that the trp aporepressor may undergo a concentration dependent association. The in vivo role of this association is not clear. However, the possibility of DNA loop formation through a protein-protein interaction is consistent with observations on several other gene regulatory proteins.
dc.format.extent 179 p.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.subjectBiochemistry
dc.title Physical examination of binding parameters for tryptophan and oligomerization of tryptophan repressor and its site-specific mutants
dc.type.genre Thesis
dc.type.material Text
thesis.degree.department Chemistry
thesis.degree.discipline Natural Sciences
thesis.degree.grantor Rice University
thesis.degree.level Doctoral
thesis.degree.name Doctor of Philosophy
dc.identifier.citation Chou, Wei-Yuan. "Physical examination of binding parameters for tryptophan and oligomerization of tryptophan repressor and its site-specific mutants." (1988) Diss., Rice University. https://hdl.handle.net/1911/16331.


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