Investigations of backbone hydrogen bonding and voltage gated mechanisms for designed gramicidin channels using backbone chemical modifications and deuterium magnetic resonance and single channel methods.

摘要

Membrane-spanning gramicidin A (gA) channels are formed by the N-terminal association of two gA monomers with the sequence formyl-VGAL&barbelow;AV&barbelow;VV&barbelow;WL&barbelow;WL&barbelow;WL&barbelow;W-ethanolamine (D amino acids are underlined). The cation-selective pore is lined by the peptide backbone. Structure and function of gA analogues with peptide backbone modifications at the join of the two monomers were analyzed.; To investigate the role of the carbonyl and amide moieties for ion channel structure and function, selected peptide bonds within gramicidin were modified. Replacement of an entire peptide bond with an ethylene group between Gly 2-Ala3, Ala3-Leu4 or Leu4-Ala5 tends to abolish or greatly diminish gramicidin channel activity. In contrast, the replacement of the peptide amide bond between Val1 and Gly2 of gA by an ester or a ketomethylene (NH → CH₂) substitution has a less dramatic effect on channel properties. Interestingly, the insertion of one, two, and even three -CH₂- groups at position 2 in the gA backbone is tolerated.; Introducing a chiral mismatch in the form of consecutive L-Ala residues at the subunit interface results in gA analogues that exhibit voltage-dependent gating. Previously characterized voltage-dependent channels have been heterodimers, thus making structural analysis difficult. A new gramicidin, (des-Val 1)-[L-Ala2]gA, displays a distribution between low and high-conductance states—depending on the applied potential—as a homodimer. The gating transitions are thought to be induced by a stress on the peptide backbone. This stress results from the chirality mismatch at position 2 due to the presence of L-Ala rather than D-Ala. In order to examine the structural basis behind the gating behavior, the side chain and chirality at position 2 were varied by incorporating L-Ala, D-Ala, Gly or aminoisobutyric acid (Alb) at this position. Analogues were deuterium-labeled using L-Ala-d ₄ or L-Val-d₈ at position 3, allowing the direct examination of local changes in the structure using solid state deuterium NMR. Deuterium NMR spectra of these peptides indicate multiple conformations for the L-Ala and Aib analogues, while spectra for the other two analogues are consistent with a single conformation. Methods were explored for recording deuterium NMR spectra while applying a variable electric field across aligned membrane samples.