The role of proline and glycine in determining the backbone flexibility of a channel-forming peptide.
Alamethicin is a helical 20-amino acid voltage-gated channel-forming peptide, which is known to exhibit segmental flexibility in solution along its backbone near alpha-methylalanine (MeA)-10 and Gly-11. In an alpha-helical configuration, MeA at position 10 would normally hydrogen-bond with position 14, but the presence of proline at this position prevents the formation of this interhelical hydrogen bond. To determine whether the presence of proline at position 14 contributes to the flexibility of this helix, two analogs of alamethicin were synthesized, one with proline 14 replaced by alanine and another with both proline 14 and glycine 11 replaced by alanine. The C-termini of these peptides were derivatized with a proxyl nitroxide, and paramagnetic enhancements produced by the nitroxide on the Calpha protons were used to estimate r-6 weighted distances between the nitroxide and the backbone protons. When compared to native alamethicin, the analog lacking proline 14 exhibited similar C-terminal to Calpha proton distances, indicating that substitution of proline alone does not alter the flexibility of this helix; however, the subsequent removal of glycine 11 resulted in a significant increase in the averaged distances between the C- and N-termini. Thus, the G-X-X-P motif found in alamethicin appears to be largely responsible for mediating high-amplitude bending motions that have been observed in the central helical domain of alamethicin in methanol. To determine whether these substitutions alter the channel behavior of alamethicin, the macroscopic and single-channel currents produced by these analogs were compared. Although the substitution of the G-X-X-P motif produces channels with altered characteristics, this motif is not essential to achieve voltage-dependent gating or alamethicin-like behavior.
Department of Chemistry and Biophysics Program at the University of Virginia, Charlottesville, Virginia 22901 USA.
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Jacob
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Duclohier
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Cafiso
D000408:Alamethicin / Q000031:analogs & derivatives / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D004578:Electron Spin Resonance Spectroscopy; D005998:Glycine / Q000737:chemistry*; D006860:Hydrogen Bonding; D007473:Ion Channels / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011392:Proline / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013113:Spin Labels
D007473:Ion Channels; D013113:Spin Labels; D000408:Alamethicin; D011392:Proline; D005998:Glycine