Structure-function relationships in helix-bundle channels probed via total chemical synthesis of alamethicin dimers: effects of a Gln7 to Asn7 mutation.
Alamethicin channels are prototypical helix bundles that may serve as tractable models for more complex protein ion channels. Solid-phase peptide synthesis of alamethicin analogues using FMOC-amino acid fluorides followed by chemical dimerization of these peptides facilitates structure-function studies of particular channel states in bilayer membranes. State 3 in particular, tentatively assigned to a hexameric helix bundle, is sufficiently long-lived that current-voltage measurements can be made during the lifetime of an individual channel opening. Molecular models of hexameric helix bundles, generated using restrained molecular dynamics with simulated annealing, indicate that a Gln7-->Asn7 (Q7-->N7) mutation will increase channel diameter locally. Experimentally, the conductance of state 3 of the N7-alm channel is found to be larger than that of the Q7-alm channel when ion flow is in the usual direction (cations entering the C-terminal end of the channel). When ion flow is in the opposite direction, no difference in the conductances of state 3 of Q7 and state 3 of N7 channels is observed. These results indicate that the effect of a change in pore diameter at position 7 is dependent on the magnitude of other barriers to permeation and that these barriers are voltage-dependent.
Jaikaran
D C
DC
Department of Chemistry, University of Toronto, Canada.
Biggin
P C
PC
Wenschuh
H
H
Sansom
M S
MS
Woolley
G A
GA
D000408:Alamethicin / Q000138:chemical synthesis* / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics*; D019281:Dimerization; D004553:Electric Conductivity; D005973:Glutamine / Q000235:genetics*; D007473:Ion Channels / Q000138:chemical synthesis* / Q000235:genetics / Q000502:physiology; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D007473:Ion Channels; D010455:Peptides; D005973:Glutamine; D000408:Alamethicin; D001216:Asparagine