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

Ln an effort to quantify the importance of hydrogen bonding and alpha-helix formation to protein stability, a capping box motif was introduced into th e small phosphocarrier protein HPr. Previous studies had confirmed that Ser 46, at the N-cap position of the short helix-B in HPr, serves as an N-cap i n solution. Thus, only a single-site mutation was required to produce a can onical 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 re sulting K49E HPr show that the designed variant is approximate to 2 kcal mo l(-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 heli x-B to the four-stranded beta-sheet of HPr. Here, we demonstrate that a str ategy in which new non-native H-bonds are introduced can generate proteins with increased stability. We discuss why the original earring box design fa iled, 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. (C) 1999 Academic Press.