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A study of the structure and function of the Escherichia coli aspartate receptor c-fragment
Abstract
The cytoplasmic region (c-fragment) of the E. coli aspartate receptor plays an important role in modulating intracellular signaling cascade and adaptation. This study focuses on understanding the structural organization of the receptor and characterizing the interactions between the receptor and the methyltransferase. Monitored by circular dichroism (CD), thermal denaturation of the c-fragment was found to be reversible. The c-fragment derived from signaling mutants displayed a second low-temperature transition that correlated with the disappearance of the oligomeric form in gel filtration chromatography (GFC). An analysis of secondary structure indicated that the oligomers were more folded than monomers. A nondenaturing detergent, octylglucoside (OG), and an $\alpha$-helix promoting reagent, trifluoroethanol (TFE), were used to reveal perturbing influences on the secondary and tertiary structure of the c-fragment and were interpreted to influence the extent of coiled-coil in the c-fragment. OG was found to diminish the cooperativity of the thermal denaturation, which could be restored by the addition of glycerol and phospholipid. These conditions where the cooperatlve transition was observed correlated with the conditions where methylation of the intact receptor had been observed. Binding between the receptor and the methyltransferase was characterized using isothermal titration calorimetry (ITC), giving a complex of 1:1 stoichiometry. Both dimeric and monomeric protein behaved in the same way, suggesting that the interaction between the receptor and the methyltransferase was not influenced by an equilibrium between the monomer and dimer of the cytoplasmic domain. The c-fragment of the receptor proved to have thie same binding activity as did the full-length receptor; a deletion of either 36 or 16 amino acids from the carboxyl terminus led to a csmplete loss of the binding activity. Finally, a synthetic peptide corresponding to the last five amino acids (NWETF) was found to have all binding activity of the full-length receptor. Evidence that the NWETF binding site for the transferase, which is strictly conserved among the serine and aspartate receptors, has physiological significance was obtained in assays of the transferase activity in which excess peptide was able to completely block the receptor methylation. The binding site was thus identified with the carboxyl "tail" of the receptor, indicating the mechanism by which the transferase can modify all the sites of methylation which it is bound to the receptor. Also, methylation between receptors was deduced as a possible consequence of this mode of binding, by comparing the data obtained in this study with numerous observations on receptor methylation and adaptation found in literature.
Subject Area
Biochemistry|Biophysics|Molecular biology
Recommended Citation
Wu, Jiongru, "A study of the structure and function of the Escherichia coli aspartate receptor c-fragment" (1996). Doctoral Dissertations Available from Proquest. AAI9639052.
https://scholarworks.umass.edu/dissertations/AAI9639052