INVESTIGATION OF A SIDE-CHAIN SIDE-CHAIN HYDROGEN-BOND BY MUTAGENESIS, THERMODYNAMICS, AND NMR-SPECTROSCOPY

Anomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine-containing protein (HPr) from two microor ganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the prop osal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an in teraction, a mutant in which Ser 31 is replaced by an alanine was gene rated in HPr from Escherichia coli. The activity, stability, and struc ture of the mutant HPr were assessed using a reconstituted assay syste m, analysis of solvent denaturation curves, and NMR, respectively. Sub stitution of Ser 31 yields a fully functional protein that is only sli ghtly less stable (Delta Delta G(folding) = 0.46 +/- 0.15 kcal mol(-1) ) than the wild type. The NMR results confirm the identity of the hydr ogen bond acceptor as Asp 69 and reveal that it exists as the gauche(- ) conformer in wild-type HPr in solution but exhibits conformational a veraging in the mutant protein. The side chain of Asp 69 interacts wit h two main-chain amide protons in addition to its interaction with the side chain of Ser 31 in the wild-type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network o f interactions. A complete analysis of the thermodynamics was performe d in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for. This analysis, pe rformed in the context of information afforded by the NMR studies, ind icates that this network of interactions contributes ca. 4-5 kcal mol( -1) to the conformational free energy of wild-type HPr.