This paper describes the effect of N-capping substitutions on the structure and stability of histidine-containing protein (HPr). We have used NMR spectroscopy and conformational stability studies to quantify changes 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 of 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 (Gram-negative) also makes it a poor substrate in the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Therefore, to understand the mechanism of inactivation of E. coli S46D HPr, as well as the effect of mutagenesis on protein stability, we have characterized three mutants 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 Asp > Asn > Ser > Ala. In addition, since there are neither large-scale conformational changes nor detectable changes in the active site of S46D HPr, it is proposed that the loss of phosphotransfer activity of S46D HPr is due to unfavorable steric and/or electrostatic interactions of the Asp with enzyme I of the PTS. Study holds ProTherm entries: 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 14019, 14020, 14021 Extra Details: Escherichia coli HPr; conformational stability; free energy;,electrostatic interactions; helix; histidine
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:18 p.m.
|Number of data points||11|
|Proteins||Phosphocarrier protein HPr ; Phosphocarrier protein HPr ; Phosphocarrier protein HPr ; Phosphocarrier protein HPr|
|Assays/Quantities/Protocols||Experimental Assay: ddG ionic:-: - ; Experimental Assay: Tm ; Experimental Assay: ddG ionic::|
|Libraries||Mutations for sequence MFQQEVTITAPNGLHTRPAAQFVKEAKGFTSEITVTSNGKSASAKSLFKLQTLGLTQGTVVTISAEGEDEQKAVEHLVKLMAELE|