Peptide adsorption to lipid bilayers: slow processes revealed by linear dichroism spectroscopy

Abstract

The adsorption and insertion kinetics for the association of two 34-residue cyclic peptides with phosphocholine membranes have been studied using circular and linear dichroism approaches. The two peptides studied are identical with the exception of two residues, which are both tyrosine in one of the peptides and tryptophan in the other. Both peptides adopt random coil conformations in solution in the absence of membranes and do not aggregate at concentrations below 20 microM. After addition to liposome dispersions, circular dichroism spectroscopy indicated that both peptides undergo an extremely rapid transformation to a beta-conformation that remains unchanged throughout the remainder of the experiment. Linear dichroism (LD) spectroscopy was used to study the kinetics of membrane adsorption and insertion. The data were analyzed by nonlinear least squares approaches, leading to identification of a number of bound states and their corresponding LD spectra. Two pseudo-first order processes could be identified that were common to both peptides. The first occurred with a time constant of the order of 1 min and led to a bound state characterized by weak LD signals, with significant bands corresponding to the transitions of aromatic side chains. The second process occurred with an unusually long time constant of between 75 and 100 min, forming a state with considerably stronger positive LD absorbance in the far-ultraviolet region of the spectrum. For the tyrosine-substituted peptide, a third slow process with a long time constant (76 min) could also be delineated and was attributed to rearrangements of the peptide within the membrane.

Figure 1

Structures of the cyclic peptides described in this study.

Figure 2

The kinetics model used for analysis of LD data.

Figure 3

Time series spectra acquired at 20°C over an 8-h period after the addition of peptides to 100 nm EPC liposomes in 10 mM tris/150 mM NaCl at pH 7.4. CD spectra for 1 (A) and 2 (B) at respective peptide concentrations of 10.1 μM and 3.23 μM and an EPC concentration of 1.82 mg/mL (2 mM); LD spectra for 1 (C) and 2 (D) at respective peptide concentrations of 1.05 μM and 0.34 μM and an EPC concentration of 1.5 mg/mL (2 mM). The legend associated with each LD spectrum indicates the time (min) after which acquisition of the corresponding spectrum commenced.

Figure 4

The evolution of LD signals after the addition of peptides 1 and 2 at respective concentrations of 1.05 μM and 0.34 μM to EPC liposomes (1.5 mg/mL; 2 mM) in 10 mM tris/150 mM NaCl at pH 7.4: semi-log plots for peptide 1 at 193 nm (A) and 2 at 228 nm (B); experimental and calculated profiles for peptides 1 (C) and 2 (D) at 193 nm (■), 228 nm (▴), 245 nm (□) and 254 nm (○). Experimental data are presented as points and the calculated profiles from nonlinear regression as lines.

Figure 5

LD spectra for each binding state described by Eq. 1, calculated by regression analysis of the binding profiles for the association of peptides 1 (A) and 2 (B) with EPC liposomes in 10 mM tris/150 mM NaCl at pH 7.4. The identity of the binding state is indicated by the text associated with each spectrum.

Figure 6

Concentrations of each of the liposome-bound states after addition of peptides 1 (A) and 2 (B) at respective concentrations of 1.05 μM and 0.34 μM to EPC liposomes (1.5 mg/mL; 2 mM) in 10 mM tris/150 mM NaCl at pH 7.4, calculated by fitting of Eq. 1 to the experimental data by nonlinear regression. (Dashed line) [P]₁; (solid line) [P]₂; (dotted line) [P]₃.

Figure 7

Peptide binding conformations consistent with the calculated LD spectra for binding states of peptides 1 and 2. The membrane is represented by the hatched boxes and the peptide by open rectangles.