Abstract
In an effort to quantify the importance of hydrogen bonding and α-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 ≃ 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 β-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.
Original language | English (US) |
---|---|
Pages (from-to) | 1609-1619 |
Number of pages | 11 |
Journal | Journal of Molecular Biology |
Volume | 286 |
Issue number | 5 |
DOIs | |
State | Published - Mar 12 1999 |
Externally published | Yes |
Keywords
- Hydrogen bonds
- N-capping
- NMR
- Protein engineering
- Protein stability
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology