Noncontact Dipole Effects on Channel Permeation. I. Experiments with (5F-Indole)Trp13 Gramicidin A Channels

Abstract

Gramicidin A (gA), with four Trp residues per monomer, has an increased conductance compared to its Phe replacement analogs. When the dipole moment of the Trp13 side chain is increased by fluorination at indole position 5 (FgA), the conductance is expected to increase further. gA and FgA conductances to Na+, K+, and H+ were measured in planar diphytanoylphosphatidylcholine (DPhPC) or glycerylmonoolein (GMO) bilayers. In DPhPC bilayers, Na+ and K+ conductances increased upon fluorination, whereas in GMO they decreased. The low ratio in the monoglyceride bilayer was not reversed in GMO-ether bilayers, solvent-inflated or -deflated bilayers, or variable fatty acid chain monoglyceride bilayers. In both GMO and DPhPC bilayers, fluorination decreased conductance to H+ but increased conductance in the mixed solution, 1 M KCl at pH 2.0, where K+ dominates conduction. Eadie-Hofstee plot slopes suggest similar destabilization of K+ binding in both lipids. Channel lifetimes were not affected by fluorination in either lipid. These observations indicate that fluorination does not change the rotameric conformation of the side chain. The expected difference in the rate-limiting step for transport through channels in the two bilayers qualitatively explains all of the above trends.

The Preference of Tryptophan for Membrane Interfaces

“…1C); in the (misfolded) DS structures (Fig. 1D), the outer Trp 13 and Trp 15 (colored magenta in Fig. 1) retain positions close to the bilayer/solution interface, whereas the inner Trp 9 and Trp 11 (colored maroon in Fig.…”

“…Previous experiments on gA 3 suggested that the indole dipole moment (13)(14)(15)(16)(17) has a direct influence on membrane protein function (ion permeation) and that indole NH hydrogen bonding is important for the conformational preference of gA (18,19). More generally, indole N-H hydrogen bonding appears to be important for protein folding and dynamics (20).…”

“…Previously, the importance of the indole ring dipole moment has been studied using Trp 3 5-F-Trp substitutions, which enhance the magnitude of the dipole moment yet leave other Trp side-chain properties unchanged (13,15,16). In the present study, to focus on the contribution of indole hydrogen bonding, we introduce a methyl group to replace the hydrogen attached to the indole nitrogen (N-H 3 N-CH 3 ).…”

“…Using 1-Me-Trp, we synthesized singly substituted gA analogues in which Trp at either position 9, 11, 13 or 15 was replaced. To study the accumulation of effects that may accompany the loss of hydrogen bonding ability, we also made two double substitutions ("inner" pair, 1-Me-Trp 9,11 ; "outer" pair, 1-Me-Trp 13,15 ). In the SS ␤-helical channel structure, all four Trps are situated close to the bilayer/solution interface (Fig.…”

“…The properties of the pairwise methylated gA analogues could therefore become very sensitive to the "masking" of the indole-NH hydrogen bond. For completeness, we also prepared the quadruply substituted gA analogue with 1-Me-Trp 9,11,13,15 .…”