Kinetic Studies of the Interaction of β-Lactoglobulin with Model Membranes: Stopped-Flow CD and Fluorescence Studies

Abstract

β-Lactoglobulin (βLG) is a member of the lipocalin protein family and has been shown to undergo changes in structure upon interaction with various surfactants and lipid vesicles. Dynamic light scattering (DLS) and Fourier transform infrared (FTIR) were used to study the interaction of dimyristoylphosphatidylglycerol (DMPG) vesicles with βLG and showed that the vesicles increase in size and become more dispersed and the transitions become broader but the lipid IR spectra and transition temperatures are stable. Fluorescence studies with vesicles with trapped calcein demonstrate βLG binding induces leakage in DMPG vesicles. To study the dynamics of the interaction between βLG and lipid vesicles further, we undertook kinetic studies using stopped-flow methods. Following previous equilibrium studies, three lipids, DMPG, dioleoylphosphatidylglycerol, and distearoylphosphatidylglycerol, all having the same negatively charged headgroup but with different acyl chain lengths, were used to prepare vesicles. Kinetic circular dichroism and fluorescence decays were measured simultaneously to monitor changes in secondary and tertiary structures, respectively. These were fit to either single- or double-exponential functions, and a multistep model, with at least two intermediate states, has been proposed to explain the major states seen in the kinetic interaction of βLG with lipid vesicles.