Effect of structural transition of the host assembly on dynamics of an ion channel peptide: a fluorescence approach

Abstract

Structural transition can be induced in charged micelles by increasing the ionic strength of the medium. We have monitored the organization and dynamics of the functionally important tryptophan residues of gramicidin in spherical and rod-shaped sodium dodecyl sulfate micelles utilizing a combination of wavelength-selective fluorescence and related fluorescence approaches. Our results show that tryptophans in gramicidin, present in the single-stranded beta(6.3) conformation, experience slow solvent relaxation giving rise to red edge excitation shift in spherical and rod-shaped micelles. In addition, changes in fluorescence polarization with increasing excitation or emission wavelength reinforce that the gramicidin tryptophans are localized in motionally restricted regions of these micelles. Fluorescence quenching experiments using acrylamide as a quencher of tryptophan fluorescence show that there is reduced water penetration in rod-shaped micelles. Taken together, we show that gramicidin conformation and dynamics is sensitive to the salt-induced structural transition in charged micelles. In addition, these results demonstrate that deformation of the host assembly could modulate protein conformation and dynamics.

FIGURE 1

Far-UV CD spectra of gramicidin in SDS micelles in the absence (solid line) and presence (dashed line) of 0.5 M NaCl. The ratio of gramicidin/SDS was 1:500 (mol/mol) and the concentration of gramicidin was 32 μM. See Materials and Methods for other details. The inset shows a schematic representation of the sphere-to-rod transition in charged SDS micelles induced by salt. Note that the headgroup spacing is reduced in rod-shaped micelles due to attenuation of interactions among the charged headgroups by the added salt.

FIGURE 2

Fluorescence emission spectra of gramicidin in SDS micelles in the absence (solid line) and presence (dashed line) of 0.5 M NaCl. The excitation wavelength used was 280 nm. All other conditions are as in Fig. 1. See Materials and Methods for other details.

FIGURE 3

Effect of changing excitation wavelength on the wavelength of maximum emission of gramicidin in SDS micelles in the absence (○) and presence (•) of 0.5 M NaCl. All other conditions are as in Fig. 1. See Materials and Methods for other details.

FIGURE 4

Fluorescence polarization of gramicidin in SDS micelles in the absence (○) and presence (•) of 0.5 M NaCl as a function of excitation wavelength. The emission wavelength was 331 nm in all cases. The data points shown are the means ± SE of at least three independent measurements. All other conditions are as in Fig. 1. See Materials and Methods for other details.

FIGURE 5

Fluorescence polarization of gramicidin in SDS micelles in the absence (○) and presence (•) of 0.5 M NaCl as a function of emission wavelength. The excitation wavelength was 280 nm in all cases. The data points shown are the means ± SE of at least three independent measurements. All other conditions are as in Fig. 1. See Materials and Methods for other details.

FIGURE 6

Time-resolved fluorescence intensity decay of gramicidin in spherical SDS micelles. Excitation wavelength was at 297 nm, which corresponds to a peak in the spectral output of the nitrogen lamp. Emission was monitored at 340 nm. The sharp peak on the left is the lamp profile. The relatively broad peak on the right is the decay profile, fitted to a biexponential function. The two bottom plots show the weighted residuals and the autocorrelation function of the weighted residuals. All other conditions are as in Fig. 1. See Materials and Methods for other details.

FIGURE 7

Representative data for Stern-Volmer analysis of acrylamide quenching of gramicidin fluorescence in SDS micelles in the absence (○) and presence (•) of 0.5 M NaCl. F_o is the fluorescence in the absence of quencher, and F is the corrected fluorescence in the presence of quencher. The excitation wavelength was fixed at 295 nm and emission was monitored at 334 nm. Concentration of SDS was 16 mM and the ratio of gramicidin/SDS was 1:4000 (mol/mol). See Materials and Methods for other details.