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Seeking the primary sense: A biochemical and biophysical study of the signaling mechanism of bacterial chemotaxis

Jiayin Li, University of Massachusetts Amherst


Genetic, biochemical, and biophysical methods have been adopted to characterize the interactions between ligand and transmembrane receptor, between receptor and cytoplasmic proteins, and between cytoplasmic proteins in the bacterial chemosensory system in order to elucidate the signaling mechanism at the molecular level.^ Ligand binding to the serine receptor (Tsr) of Escherichia coli has been studied using Isothermal Titration Calorimetry (ITC) to determine the number of moles of ligand bound per mole of receptor (n), the binding constant $(K\sb{\rm a}),$ and the enthalpy of binding $(\Delta H)$ of serine to the receptor. The n-value for serine binding to octyl-$\beta$-D-glucopyranoside (OG)-solubilized Tsr (n = 0.5) was consistent with one molecule of serine binding to a receptor dimer. The values for $K\sb{\rm a}$ and $\Delta H$ were equivalent for the membrane and OG-solubilized samples and were found to be $\rm 3.6\times 10\sp4\ M\sp{-1}$ and $-18$ kcal/mol at 27$\sp\circ$C. Covalent modification of Tsr at the sites of methylation was found to have only a modest effect on serine binding.^ The interaction between Tsr and the methyltransferase (CheR) was also studied using ITC. Tsr bound to CheR with 1 to 1 ratio and a dissociation constant of 2 $\mu$M at 29$\sp\circ$C. Ligand-binding or modifications at the methylation sites of Tsr did not affect CheR binding. Truncation of Tar cytoplasmic-fragment at the C-terminus by 16 amino acids (aa) completely abolished CheR binding. This result led to the identification of the CheR binding site which is located at the C-terminal 5-aa segment remote from the methylation regions. The C-terminal truncated and crosslinkable Tsr mutant ($\Delta$34, D36C) was then constructed, which was found to be a poor substrate for CheR by itself. Coexpression of this binding site deleted Tsr with intact Tsr rescued its methylation activity even in the crosslinked form. The mechanism of interdimer methylation was established through this study.^ The autophosphorylating kinase CheA donates a phosphoryl group to either of two response regulator proteins, CheY or CheB. With ITC, it was demonstrated that CheA and a CheA fragment composed of aa residues 1 to 233 (CheA$\sb{1-233}$ bound to CheY with similar dissociation constants of 2.0 and 1.2 $\mu$M at 28$\sp\circ$C, respectively, indicating that the CheY binding site is wholly within the 1-233 aa locus. CheB bound to CheA$\sb{1-233}$ with a $K\sb{\rm d}$ of 3.2 $\mu$M, and also bound to intact CheA with the same affinity. CheY was found to compete with CheB for binding to CheA$\sb{1-233}.$ Phosphorylated CheY, in the presence of 6 mM Mg$\sp{2+},$ has a significantly lower affinity for CheA (20% of unphosphorylated form), but mutations at the active site of CheY have little effect on CheY-CheA interaction, a mutation remote from the active site (A103V) produced a 10-fold reduction in $K\sb{\rm a},$ indicating the separation of the binding site from the active site and a significant conformational change upon phosphorylation. ^

Subject Area

Molecular biology|Cellular biology|Biochemistry

Recommended Citation

Li, Jiayin, "Seeking the primary sense: A biochemical and biophysical study of the signaling mechanism of bacterial chemotaxis" (1996). Doctoral Dissertations Available from Proquest. AAI9709621.