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


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 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