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
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:31 p.m.
|Number of data points||11|
|Proteins||Phosphocarrier protein HPr ; Phosphocarrier protein HPr|
|Assays/Quantities/Protocols||Experimental Assay: dCp ; Experimental Assay: ddG ionic:-: -, temp:63.4 C ; Experimental Assay: ddG temp:30.0 C, ionic:: ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence MFQQEVTITAPNGLHTRPAAQFVKEAKGFTSEITVTSNGKSASAKSLFKLQTLGLTQGTVVTISAEGEDEQKAVEHLVKLMAELE|