Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy.


Abstract

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 PDB Entries

Structure ID Release Date Resolution Structure Title
1CM2 1999-05-13T00:00:00+0000 1.8 STRUCTURE OF HIS15ASP HPR AFTER HYDROLYSIS OF RINGED SPECIES.
1CM3 1999-05-13T00:00:00+0000 1.6 HIS15ASP HPR FROM E. COLI
1GGR 2000-09-18T00:00:00+0000 0 COMPLEX OF ENZYME IIAGLC AND THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA COLI NMR, RESTRAINED REGULARIZED MEAN STRUCTURE
1HDN 1994-02-10T00:00:00+0000 0 THE HIGH-RESOLUTION STRUCTURE OF THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA COLI DETERMINED BY RESTRAINED MOLECULAR DYNAMICS FROM NMR NUCLEAR OVERHAUSER EFFECT DATA
1J6T 2002-08-14T00:00:00+0000 0 COMPLEX OF ENZYME IIAMTL AND THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA COLI NMR, RESTRAINED REGULARIZED MEAN STRUCTURE
1OPD 1996-08-01T00:00:00+0000 1.5 HISTIDINE-CONTAINING PROTEIN (HPR), MUTANT WITH SER 46 REPLACED BY ASP (S46D)
1PFH 1995-08-18T00:00:00+0000 0 THE PHOSPHORYLATED FORM OF THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR
1POH 1993-10-19T00:00:00+0000 2.0 THE 2.0 ANGSTROMS RESOLUTION STRUCTURE OF ESCHERICHIA COLI HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR: A REDETERMINATION
1VRC 2005-02-21T00:00:00+0000 0 Complex of enzyme IIAmannose and the histidine-containing phosphocarrier protein HPr from escherichia coli nmr, restrained regularized mean structure
2JEL 1998-02-24T00:00:00+0000 2.5 JEL42 FAB/HPR COMPLEX

Relevant UniProtKB Entries

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