Conformational characterization of a helix-nucleated bicyclic GCN4 decapeptide by proton NMR

A bicyclic decapeptide, GCN4brM1, which was designed to be a helix-locked a nalog of the DNA-binding basic region from the yeast transcription factor G CN4, was synthesized and characterized using circular dichroism (CD) spectr opolarimetry and H-1-NMR. This peptide has two Lys', Asp(i+4) side chain la ctam bridges incorporated into its structure in overlapping positions in th e peptide chain, linking residues 3 and 7 and residues 4 and 8. CD spectra of GCN4brM1 in aqueous solution are consistent with the expected helical co nformation, and indicate that this conformation is remarkably resistant to heat denaturation and is essentially unchanged by addition of 50% (v/v) tri fluoroethanol (TFE) as cosolvent, NMR spectra measured in aqueous solution at -5 degrees C show long-range nuclear Overhauser effects (NOEs) that are consistent with an alpha-helical conformation throughout the peptide struct ure. The measured (3)J(HN) coupling constants are also in agreement with an alpha-helical structure. Extremely slow proton-deuterium exchange rates me asured for backbone amides in the middle of the peptide indicate that this helix is highly stabilized and rarely unfolds within the side chain bridged sequence. NOE-constrained molecular dynamics simulations gave rise to a si ngle family of converged structures that are fully alpha-helical throughout the GCN4brM1 backbone, and show a single, well-defined con-formation for t he two side chain bridges. This study demonstrates that two overlapping Lys ', Asp(i+4) lactam bridges, positioned in consecutive residue positions in a hexapeptide segment, form a rigid alpha-helical structure in aqueous solu tion that is propagated in both the N-terminal and C-terminal directions.