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Characterization of the role of side-chain interactions in the binding of ligands to apo trp repressor: pH dependence studies.

Biophysical chemistry (1997-05-21)
M Kavanoor, M R Eftink
ABSTRACT

The pH dependence of the association of apo trp repressor with the series of ligands, tryptophan, tryptamine, indole propionic acid (IPA), and trans-beta-indole acrylic acid (IAA), has been studied using fluorescence titrations and isothermal titration microcalorimetry (ITC). The purpose of such a comparison of ligands and the pH dependency studies is to reveal the role played by the side-chain functional groups in the energetics of the binding of the ligands to the protein. We find that, whereas the binding of tryptamine and IPA have essentially no pH dependence between pH 6 and 10, the binding of tryptophan and IAA depends on pH. For IAA, the affinity drops between pH 6 and 10, consistent with a shift in pKa of some group on the protein from a value of pKa 7.4 to 7.9 upon binding of this ligand. The affinity of IAA also drops below pH 5, but shows saturable binding at pH 2-3, where the protein has previously been found to exist as a partially folded monomeric state. For tryptophan, the pH dependence data indicate that the equilibrium is complicated. We present a model to describe the data in which the alpha-ammonium group of tryptophan has its pKa shifted upward upon binding (i.e. preferential binding of the protonated form of this functional group) and in which the pKa of an unknown group on the protein also has its pKa increased.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
3-Indolepropionic acid, ReagentPlus®, 99%
Sigma-Aldrich
Indole-3-propionic acid, ≥99.0% (T)
Sigma-Aldrich
Indole-3-propionic acid, ≥97.0% (T)
Sigma-Aldrich
3-Indoleacrylic acid, BioReagent, ≥98% (HPLC)
Sigma-Aldrich
trans-3-Indoleacrylic acid, 98%
Sigma-Aldrich
3-Indoleacrylic acid, powder

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