Importance of indole NH hydrogen bonding in the organization and dynamics of gramicidin channels

Abstract

The linear ion channel peptide gramicidin represents an excellent model for exploring the principles underlying membrane protein structure and function, especially with respect to tryptophan residues. The tryptophan residues in gramicidin channels are crucial for the structure and function of the channel. In order to test the importance of indole hydrogen bonding for the biophysical properties of gramicidin channels, we monitored the effect of N-methylation of gramicidin tryptophans, using a combination of steady state and time-resolved fluorescence approaches along with circular dichroism spectroscopy. We show here that in the absence of hydrogen bonding ability of tryptophans, tetramethyltryptophan gramicidin (TM-gramicidin) is unable to maintain the single stranded, head-to-head dimeric channel conformation in membranes. Our results show that TM-gramicidin displays a red-shifted fluorescence emission maximum, lower red edge excitation shift (REES), and higher fluorescence intensity and lifetime, consistent with its nonchannel conformation. This is in agreement with the measured location (average depth) of the 1-methyltryptophans in TM-gramicidin using the parallax method. These results bring out the usefulness of 1-methyltryptophan as a fluorescent tool to examine the hydrogen bonding ability of tryptophans in proteins and peptides. We conclude that changes in the hydrogen bonding ability of tryptophans, along with coupled changes in peptide backbone structure induce the loss of single stranded β6.3 helical dimer conformation. These results agree with earlier results from size-exclusion chromatography and single-channel measurements for TM-gramicidin, and confirm the importance of indole hydrogen bonding for the conformation and function of ion channels and membrane proteins.

CN stretching vibration of 5-cyanotryptophan as an infrared probe of protein local environment: what determines its frequency?

“…One such example is the aforementioned M2 proton channel, wherein the N-H ends of the key Trp41 residues, according to a recent high-resolution crystal structure, 37,38 point toward the aqueous pore, leaving the benzene rings facing the hydrocarbons of the lipid. Another example is the gramicidin A proton channel, 39,40 where H-bonding interactions involving the N-H groups of several Trp residues are believed to be crucial for stabilizing the functional channel conformation. A third example is transmembrane peptides and proteins, where Trp residues are often located at the water-membrane interface with their N-H groups H-bonded to either water or a lipid headgroup.…”

Juxtamembrane tryptophans have distinct roles in defining the OmpX barrel–micelle boundary and facilitating protein–micelle association

“…Put together, our fluorescence measurements suggest that Trp140 resides in a hydrophobic environment, and is micelle solvated. Trp76, however, is rigid and in a polar, solvent‐accessible environment [37]. Although structurally similar, location in dissimilar environments affects indole interaction with the surrounding micelles, and may confer different functionalities to OmpX indoles.…”

“…Evidence for micelle solvation of Trp140 is obtained from the average lifetime (< τ >) measurements [36,37], which reveals an unusually long lifetime of 4.6 ns for OmpX:W140 (Table 1). Solvent‐accessible tryptophans undergo non‐radiative losses and possess short lifetimes [22], as seen for Trp76.…”

A Ferrocene–Tryptophan Conjugate: The Role of the Indolic Nitrogen in Supramolecular Assembly

“…Chaudhuri et al. reported the importance of indole NH hydrogen bonding in gramicidin channels, in which methylation of the indole of NH disrupts the hydrogen bonding, resulting in the loss of membrane conformation . Our group previously reported that the indole NH group can also be involved in hydrogen bonding to neighboring molecules, which is little reported in the literature .…”