Pk. Hammen et al., INVESTIGATION OF A SIDE-CHAIN SIDE-CHAIN HYDROGEN-BOND BY MUTAGENESIS, THERMODYNAMICS, AND NMR-SPECTROSCOPY, Protein science, 4(5), 1995, pp. 936-944
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.