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

¹ Loyola University Chicago
² Loyola University Medical Center

^* To whom correspondence should be addressed. E-mail: scukier{at}lumc.edu.

Submitted on March 19, 2007
Revised on April 11, 2007
Accepted on 7 May 2007

	Abstract

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 and small group 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: SS- cyclopentane diacid, RR-cyclopentane diacid, and succinic acid. Single channel proton conductances (g_H) were measured at various proton concentrations of protons ([H⁺]), and compared to previous measurements obtained in the SS- and RR- dioxolane-linked as well as in native gA channels. Replacing the SS-dioxolane by a SS-cyclopentane had no effects on the g_H - [H⁺] relationships suggesting that the constrained and continuous transition between the two gA peptides via these SS linkers is ultimately responsible for the 2-4 fold 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 RR-cyclopentane are considerably larger than in RR-dioxolane linked gA channels. One explanation would be that the electrostatic interactions between the oxygens in the dioxolane and adjacent carbonyls in the RR-dioxolane linked gA channel attenuate the rate of H⁺ transfer in the middle of the pore. Interestingly, g_H-[H⁺] relationships in the RR-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 -helical gA peptides enhances the rate of H⁺ transfer in water wires by decreasing the activation entropy of the system.

Key Words: gramicidin, proton currents, proton transfer, single channel, single channels, water wire