INFLUENCE OF N-CAP MUTATIONS ON THE STRUCTURE AND STABILITY OF ESCHERICHIA-COLI HPR

This paper describes the effect of N-capping substitutions on the stru cture and stability of histidine-containing protein(HPr). We have used NMR spectroscopy and conformational stability studies to quantify cha nges in local and global free energy due to mutagenesis at Ser46, the N-cap for helix B in HPr. Previous NMR studies suggested that helix B of Escherichia coli HPr is dynamic as judged by the rate of exchange o f amide protons with solvent, Ser46 was chosen because it is the site of regulatory phosphorylation in HPrs from Gram-positive bacteria, and mutation of this residue to an aspartic acid (S46D) in E. coli HPr (G ram-negative) also makes it a poor substrate in the bacterial phosphoe nolpyruvate: sugar phosphotransferase system. Therefore, to understand the mechanism of inactivation of E. coli S46D HPr, as well as the eff ect of mutagenesis on protein stability, we have characterized three m utants of E. coli HPr: Ser46 has been mutated to an Asp, Asn, and Ala in S46D, S46N, and S46A HPrs, respectively. The results indicate that these N-cap replacements have a marked influence on helix B stability, The effect of mutagenesis on local stability is correlated to global unfolding of HPr. The ability of amino acids to stabilize helix B is A sp > Asn > Ser > Ala. In addition, since there are neither large-scale conformational changes nor detectable changes in the active site of S 46D HPr, it is proposed that the loss of phosphotransfer activity of S 46D HPr is due to unfavorable steric and/or electrostatic interactions of the Asp with enzyme I of the PTS.