Terminal ion pairs stabilize the second β-hairpin of the B1 domain of protein G

Abstract

The effects of terminal ion pairs on the stability of a β-hairpin peptide corresponding to the C-terminal residues of the B1 domain of protein G were determined using thermal unfolding as monitored by nuclear magnetic resonance and circular dichroism spectroscopy. Molecular dynamics (MD) simulations were also performed to examine the effect of ion pairs on the structures. Eight peptides were studied including the wild type (G41) and the N-terminal modified sequences that had the first residue deleted (E42), replaced with a Lys (K41), or extended by an additional Gly (G40). Acetylated variants were made to examine the effect of removing the positive N-terminal charge on β-hairpin stability. The rank in stability determined experimentally is K41 > E42 ≈ G41 ≈ G40 > AC-K41 > Ac-E42 ≈ Ac-G41 > Ac-G40. The Tm of the K41 peptide is 12 °C higher than G41, while the T m values for the acetylated peptides are less than their unacetylated forms by more than 15 °C. NOE cross-peaks between side-chain methylene groups at the N- and C-termini and larger CαH shifts compared to random values are seen for K41. The addition of 20% methanol increases the stability in K41 and G41. The MD studies complement these results by showing that the charged N-terminus is important to stability. The type of ion pair observed varies with peptide, and when formed the simulations show that the ion pair can prevent fraying of the α-strands through electrostatic and hydrophobic contacts. Therefore, introducing favorable electrostatic interactions at the N- and C-termini can substantially enhance β-hairpin stability and help define the structure. © 2006 Wiley-Liss, Inc.