Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles |
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Authors: | Jordan J B Easton P L Hinton J F |
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Affiliation: | Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA. |
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Abstract: | 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. |
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Keywords: | gA, gramicidin A Phe11-gA, Phe11-gramicidin A d2-TFE, deuterated trifluoroethanol (CF3CD2OH) d25-SDS, deuterated sodium dodecyl sulfate DQF-COSY, double quantum filtered correlation spectroscopy TOCSY, total correlation spectroscopy NOESY, nuclear Overhauser enhancement spectroscopy DGSA, distance geometry/simulated annealing LUV, large unilamellar vesicle SDS, sodium dodecyl sulfate RMSD, root mean-square deviation. |
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