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 microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly 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 hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche- conformer in wild-type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main-chain amide proteins 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 of interactions. A complete analysis of the thermodynamics was performed in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 7271, 7272 Extra Details: conformational free energy; hydrogen bond; NMR;,protein stability; protein structure

Submission Details

ID: SfeDNz7y3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:33 p.m.

Version: 1

Publication Details
Hammen PK;Scholtz JM;Anderson JW;Waygood EB;Klevit RE,Protein Sci. (1995) Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy. PMID:7663349
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Phosphocarrier protein HPr P0AA06 PTHP_ECO57
100.0 Phosphocarrier protein HPr P0AA05 PTHP_ECOL6
100.0 Phosphocarrier protein HPr P0AA04 PTHP_ECOLI
100.0 Phosphocarrier protein HPr P0AA08 PTHP_SALTI
100.0 Phosphocarrier protein HPr P0AA07 PTHP_SALTY
100.0 Phosphocarrier protein HPr P0AA09 PTHP_SHIFL
98.8 Phosphocarrier protein HPr P16481 PTHP_KLEPN