Using two fluorescent probes to dissect the binding, insertion, and dimerization kinetics of a model membrane peptide

Abstract

Helix-helix association within a membrane environment represents one of the fundamental processes in membrane protein folding. However, studying the kinetics of such processes has been difficult because most membrane proteins are insoluble in aqueous solution. Here we present a stopped-flow fluorescence study of the membrane-interaction kinetics of a designed, water-soluble transmembrane (TM) peptide, anti-alpha(IIb), which is known to dimerize in phospholipid bilayers. We show that by using two fluorescent amino acids, tryptophan and p-cyanophenylalanine, we are able to kinetically dissect distinct phases in the peptide-membrane interaction, representing membrane binding, membrane insertion, and TM helix-helix association. Our results further show that the last process occurs on a time scale of seconds, indicating that the association of two TM helices is an intrinsically slow event.

Figure 1

Fluorescence spectra of anti-α_IIb (pink and green) and anti-α_IIb-Phe_CN (red and blue) in buffer and POPC/G vesicle solutions, as indicated. The peptide and lipid concentrations were 2.5 µM and 0.86 mg/mL, respectively. The excitation wavelengths were 290 and 240 nm for anti-α_IIb and anti-α_IIb-Phe_CN, respectively.

Figure 2

Stopped-flow kinetics of anti-α_IIb (black) and anti-α_IIb-Phe_CN (blue) upon association with POPC/G vesicles (0.86 mg/mL). In both cases, the final peptide concentration was 2.5 µM. The smooth lines are fits of these data to the model discussed in the text.