Increased helix and protein stability through the introduction of a new tertiary hydrogen bond.


Abstract

In an effort to quantify the importance of hydrogen bonding and alpha-helix formation to protein stability, a capping box motif was introduced into the small phosphocarrier protein HPr. Previous studies had confirmed that Ser46, at the N-cap position of the short helix-B in HPr, serves as an N-cap in solution. Thus, only a single-site mutation was required to produce a canonical S-X-X-E capping box: Lys49 at the N3 position was substituted with a glutamic acid residue. Thermal and chemical denaturation studies on the resulting K49E HPr show that the designed variant is approximately 2 kcal mol-1 more stable than the wild-type protein. However, NMR studies indicate that the side-chain of Glu49 does not participate in the expected capping H-bond interaction, but instead forms a new tertiary H-bond that links helix-B to the four-stranded beta-sheet of HPr. Here, we demonstrate that a strategy in which new non-native H-bonds are introduced can generate proteins with increased stability. We discuss why the original capping box design failed, and compare the energetic consequences of the new tertiary side-chain to main-chain H-bond with a local (helix-capping) side-chain to main-chain H-bond on the protein's global stability. Study holds ProTherm entries: 6098, 6099, 6100, 6101, 14296 Extra Details: N-capping; NMR; hydrogen bonds; protein engineering;,protein stability

Submission Details

ID: 3ksxXmv64

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Peterson RW;Nicholson EM;Thapar R;Klevit RE;Scholtz JM,J. Mol. Biol. (1999) Increased helix and protein stability through the introduction of a new tertiary hydrogen bond. PMID:10064718
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
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