Insight into a Random Coil Conformation and an Isolated Helix: Structural and Dynamical Characterisation of the C-Helix Peptide from Hen Lysozyme

Abstract

A 17 residue peptide corresponding to the C-helix of hen lysozyme (residues 86 to 102) has been investigated in detail to assess the factors that determine its conformation in both aqueous and trifluoroethanol (TFE) solutions. A thorough characterisation of the peptide by CD and NMR techniques under both conditions has been performed including the determination of complete NMR proton sequential assignments, and measurement of NOE effects, 3 J HNαcoupling constants, temperature coefficients and residue-specific hydrogen-exchange rates. In water, the peptide adopts a largely unstructured conformation and NMR data, particularly coupling constants and chemical shift deviations, have been shown to agree closely with predictions from a model for a random coil based on the φ,ψ distributions in a protein database. This indicates that under these conditions the intrinsic conformational preferences of the individual amino acid residues are the dominating factors that determine the population of conformers adopted. With increasing concentrations of TFE a cooperative transition to an extensively helical conformation occurs and the resultant changes in CαH chemical shifts have been shown to correlate with the changes in φ,ψ populations. Using NOE and coupling constant data for this state, an ensemble of structures has been calculated and provides a model for a helix in the absence of tertiary interactions. In this model fluctuations, which increase in amplitude towards the termini, occur about the average helical φ,ψ angles and are responsible for increasing the values of 3 J HNαcoupling constants above those anticipated for a static helix. The residue-specific rates of hydrogen exchange for the peptide in 50% TFE-d 3are consistent with such a model, the maximum protection from exchange being observed for residues in the centre of the helix.