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Ligand binding to the serine receptor of Escherichia coli has been studied using isothermal titration calorimetry. Bacterial inner membranes enriched in the serine receptor (Tsr) were titrated as sonicated membrane samples and after solubilization in octyl beta-D-glucopyranoside (OG) to determine the number of moles of ligand bound per mole of receptor (n), the binding constant (Ka), and the enthalpy of binding (delta H) of serine to the receptor. The n value for serine binding to OG-solubilized Tsr protein (n = 0.5) was consistent with one molecule of serine binding to a receptor dimer, but in sonicated inner membrane samples, the n value was smaller (n approximately equal to 0.25), indicating that not all of the binding sites were accessible to added serine. At 7 and 27 degrees C, the values for Ka and delta H were equivalent for the membrane and OG-solubilized samples and were found to be 4.7 x 10(4) M-1 and -15 kcal/mol, and 3.6 x 10(4) M-1 and -18 kcal/mol, respectively. The influence of covalent modification at the sites of methylation on the affinity of the receptor for serine was also investigated, and found to have only a modest effect. The property of half-site saturation is suggestive of models for transmembrane signaling where the receptor subunit interactions are modulated by ligand binding.

It was suggested earlier that the slowest step normally observed in protein folding kinetics might be due to cis-trans isomerization of the peptide bonds which are N terminal to proline residues. As a means of further testing this hypothesis, the kinetics of three forms of carp parvalbumins have been examined. Two of these (bands 3 and 5) have no proline residues, while the other (band 2) has a single proline. Other than this, the amino acid sequences show a large degree of homology. The unfolding and refolding of each form have been examined under conditions where circular dichroism and fluorescence measurements suggest that the structure of the three native forms are exceedingly similar, as are the structures of the three denatured forms. In spite of the large similarity in the sequence and structure of these proteins, the kinetic