Conformational characterization of a helix-nucleated bicyclic GCN4 decapeptide by proton NMR.
A bicyclic decapeptide, GCN4brM1, which was designed to be a helix-locked analog of the DNA-binding basic region from the yeast transcription factor GCN4, was synthesized and characterized using circular dichroism (CD) spectropolarimetry and 1H-NMR. This peptide has two Lys(i), Asp(i+4) side chain lactam bridges incorporated into its structure in overlapping positions in the 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 conformation, and indicate that this conformation is remarkably resistant to heat denaturation and is essentially unchanged by addition of 50% (v/v) trifluoroethanol (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 structure. The measured 3J(HN) coupling constants are also in agreement with an alpha-helical structure. Extremely slow proton-deuterium exchange rates measured 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 single family of converged structures that are fully alpha-helical throughout the GCN4brM1 backbone, and show a single, well-defined conformation for the two side chain bridges. This study demonstrates that two overlapping Lys(i), Asp(i+4) lactam bridges, positioned in consecutive residue positions in a hexapeptide segment, form a rigid alpha-helical structure in aqueous solution that is propagated in both the N-terminal and C-terminal directions.
Zhang
M
M
Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854, USA.
Wu
B
B
Baum
J
J
Taylor
J W
JW
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D003903:Deuterium / Q000737:chemistry; D005656:Fungal Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases / Q000138:chemical synthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008024:Ligands; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; D006859:Hydrogen; D004247:DNA; D003903:Deuterium; D011494:Protein Kinases