Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles.
The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of Trp-->Phe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two beta6.3 helices joined by six hydrogen bonds at their NH2-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.
Jordan
J B
JB
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Easton
P L
PL
Hinton
J F
JF
D020816:Amino Acid Motifs; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D019281:Dimerization; D004594:Electrophysiology; D019277:Entropy; D006096:Gramicidin / Q000737:chemistry* / Q000494:pharmacology; D006358:Hot Temperature; D007477:Ions; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008958:Models, Molecular; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics*; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D013816:Thermodynamics
D000900:Anti-Bacterial Agents; D007477:Ions; D008051:Lipid Bilayers; D008823:Micelles; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D006096:Gramicidin; D012967:Sodium Dodecyl Sulfate; D010649:Phenylalanine