The effects of single amino acid replacements on the structure and function of the gramicidin A ion channel determined using nuclear magnetic resonance spectroscopy and single-channel conductance measurements.

摘要

Gramicidin is a 15 amino acid, transmembrane channel-forming peptide that is selective for monovalent cations. The parent molecule, gramicidin A, exhibits alternating L and D amino acids (LV-G-L A-DL-LA-DV-LV- DV-LW-DL-LW-D L-LW-DL-LW) and is capped by formyl and ethanolamine groups at its N- and C-terminus, respectively. Gramicidin is a commonly used model in the study of the structure and function of ion channels. Due to its small size and well-known characteristics, gramicidin is easily modified by amino acid replacement.; The structural heterogeneity amino acid substituted gramicidin analogs has been monitored using NMR spectroscopy. It has been determined that replacement of the "spacer" residues that separate the Trps has a large effect on the overall structure and stability of the channel. This is evidenced by the existence of multiple helical forms of the peptides when incorporated into SDS micelles.; The structures of phenylalanine and glycine analogs of gramicidin A have also been determined in which the Trp residues at positions 11, 13, and 15 have been replaced by phenylalanine and glycine (Phe15-, Phe 13-, Phe11-, Gly15-, Gly13-, and Gly 11-gA). The single-channel conductances and average channel lifetimes of these analogs have been measured. It is concluded that the replacement of Trp residues at these positions cause little or no effect on channel structure, but that modulation of long range ion-dipole interactions exhibit significant effects on channel function.; Lastly, the three-dimensional structure of an N-terminal modified gramicidin (des-Val1-[DAla2-LVal3]-gA) has been determined. The results confirm that, regardless of the reversal of stereochemistry at the 1 position, the channel structure formed by the 14Mer still exhibits a right-handed, beta helical structure. The single-channel conductance and average channel lifetime have been measured for this analog and indicate that while channel function is impaired, a significantly longer channel lifetime results. The determination of the three-dimensional structure of this analog indicates that this is due to the formation of an additional inter-molecular hydrogen bond at the dimer interface of the channel.