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Proton Transfer in Water Wires in Proteins: Modulation by Local Constraint and Polarity in Gramicidin A Channels

Shasikala Narayan ^*, Debra L. Wyatt , David S. Crumrine ^* and Samuel Cukierman 

^* Department of Chemistry, Loyola University Chicago, Chicago, Illinois 60626; and Department of Physiology, Loyola University Medical Center, Maywood, Illinois, 60153

Correspondence: Address reprint requests to Dr. Samuel Cukierman, Dept. of Physiology, Loyola University Medical Center, 2160 South First Ave., Maywood, IL 60153. E-mail: scukier{at}lumc.edu.

The transfer of protons in membrane proteins is an essential phenomenon in biology. However, the basic rules by which H⁺ transfer occurs in water wires inside proteins are not well characterized. In particular, the effects of specific atoms and small groups of atoms on the rate of H⁺ transfer in water wires are not known. In this study, new covalently linked gramicidin-A (gA) peptides were synthesized, and the effects of specific atoms and peptide constraints on the rate of H⁺ transfer were measured in single molecules. The N-termini of two gA peptides were linked to various molecules: S,S-cyclopentane diacid, R,R-cyclopentane diacid, and succinic acid. Single-channel proton conductances (g_H) were measured at various proton concentrations ([H⁺]) and compared to previous measurements obtained in the S,S- and R,R-dioxolane-linked as well as in native gA channels. Replacing the S,S-dioxolane by an S,S-cyclopentane had no effects on the g_H-[H⁺] relationships, suggesting that the constrained and continuous transition between the two gA peptides via these S,S linkers is ultimately responsible for the two- to fourfold increase in g_H relative to native gA channels. It is likely that constraining a continuous transition between the two gA peptides enhances the rate of H⁺ transfer in water wires by decreasing the number of water wire configurations that do not transfer H⁺ at higher rates as in native gA channels (a decrease in the activation entropy of the system). On the other hand, g_H values in the R,R-cyclopentane are considerably larger than those in R,R-dioxolane-linked gA channels. One explanation would be that the electrostatic interactions between the oxygens in the dioxolane and adjacent carbonyls in the R,R-dioxolane-linked gA channel attenuate the rate of H⁺ transfer in the middle of the pore. Interestingly, g_H-[H⁺] relationships in the R,R-cyclopentane-linked gA channel are quite similar to those in native gA channels. g_H values in succinyl-linked gA channels display a wide distribution of values that is well represented by a bigaussian. The larger peaks of these distributions are similar to g_H values measured in native gA channel. This observation is also consistent with the notion that constraining the transition between the two ß-helical gA peptides enhances the rate of H⁺ transfer in water wires by decreasing the activation entropy of the system.