Monitoring Gramicidin Conformations in Membranes: A Fluorescence Approach

Abstract

We have monitored the membrane-bound channel and nonchannel conformations of gramicidin utilizing red-edge excitation shift (REES), and related fluorescence parameters. In particular, we have used fluorescence lifetime, polarization, quenching, chemical modification, and membrane penetration depth analysis in addition to REES measurements to distinguish these two conformations. Our results show that REES of gramicidin tryptophans can be effectively used to distinguish conformations of membrane-bound gramicidin. The interfacially localized tryptophans in the channel conformation display REES of 7 nm whereas the tryptophans in the nonchannel conformation exhibit REES of 2 nm which highlights the difference in their average environments in terms of localization in the membrane. This is supported by tryptophan penetration depth measurements using the parallax method and fluorescence lifetime and polarization measurements. Further differences in the average tryptophan microenvironments in the two conformations are brought out by fluorescence quenching experiments using acrylamide and chemical modification of the tryptophans by N-bromosuccinimide. In summary, we report novel fluorescence-based approaches to monitor conformations of this important ion channel peptide. Our results offer vital information on the organization and dynamics of the functionally important tryptophan residues in gramicidin.

Novel Insights Into Protein Structure and Dynamics Utilizing the Red Edge Excitation Shift Approach

“…60,66 The results from these studies point out the motional restriction experienced by the tryptophans at the peptide-lipid interface of the gramicidin channel. 153,156 This is consistent with other studies 60,66 in which such a restriction is thought to be imposed due to hydrogen bonding between the indole rings of the tryptophan residues in the channel conformation and the neighboring lipid carbonyls. The significance of such organization in terms of functioning of the channel is brought out by the fact that substitution, photodamage, or chemical modification of these tryptophans are known to give rise to channels with altered conformation and reduced conductivity.…”

“…In another study, the magnitude of REES of membrane-bound melittin has been shown to be dependent on the extent of lipid chain unsaturation in the membrane. It is interesting to note that the magnitude of REES obtained with melittin in membranes containing more than two double bonds is considerably higher (up to 19 nm) 152 than what is usually reported for membrane-bound tryptophan residues, 62,146,147,[153][154][155][156] although higher REES has been reported in a few cases (see below). In addition, the environment of tryptophan residues of several tryptophan-rich antimicrobial peptides in the membrane-bound form has been explored utilizing REES.…”

“…In addition, REES has been used to monitor the effects of structural transition of the host assembly and hydration on the organization and dynamics of gramicidin. 164,165 Further, REES of gramicidin has been found to be dependent on the lipid composition 166 and phase state of the membrane. 167 In the absence of crystal structures, fluorescence approaches to study the topography and membrane interactions of specific peptide fragments have proved to be very useful.…”

“…157 The phenomenon of REES, in conjunction with time-resolved fluorescence spectroscopic parameters such as wavelength-dependent fluorescence lifetimes and timeresolved emission spectra (TRES) was utilized to probe the localization and dynamics of the functionally important tryptophan residues in the gramicidin channel. 153,156 Gramicidin belongs to a family of prototypical cation-selective channel formers, which are naturally fluorescent due to the presence of four tryptophan residues. 158 These interfacially localized tryptophans are known to play a crucial role in the organization and function of the channel.…”

“…60,66,[159][160][161][162][163] More importantly, REES and related fluorescence approaches have been used to distinguish between the channel and non-channel conformations of gramicidin in membranes. 156 REES of gramicidin is therefore sensitive to the conformation adopted by the peptide in the membrane. This provides a convenient spectroscopic handle to monitor the functional status of this important ion channel peptide.…”