The Phosphoenolpyruvate-Dependent Phosphotransferase System of Staphylococcus aureus. 3. 1H and 31P Nuclear-Magnetic-Resonance Studies on the Phosphocarrier Protein HPr; Tyrosine Titration and Denaturation Studies

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Abstract
The phosphocarrier protein HPr was investigated by proton NMR at 270 MHz to evaluate structural properties of the whole molecule and its active site. The titration behavior of the 3 tyrosines of the HPr protein was analyzed by monitoring the chemical shifts of the aromatic proton resonances of these residues as a function of pH. The HPr protein contains a lot of slowly exchanging NH backbone protons which suggested a relatively rigid secondary structure of the protein molecule itself although it contains no disulfide bridges. The HPr protein shows a sharp reversible denaturation behavior at alkaline pH values. Between pH 10.8-11.1, 2 C-2 proton resonance peaks for the single histidine residue could be observed together with abrupt changes in the aromatic and aliphatic absorption region of the HPr protein which are due to chemical exchange processes. The NMR spectrum of the HPr protein is only changed a little on raising the temperature from 14.degree. C to 70.degree. C. At 76.degree. C all resonances in the spectrum broaden and almost disappear. This process is irreversible.