STRUCTURE OF b-LACTOGLOBULIN INTERACTING WITH LIPID VSEICLES: A SPECTROSCOPIC AND THERMODYNAMIC STUDY

Ge, Ning

SUMMARY

The work in this thesis focused on the optical spectroscopic, thermodynamic and kinetic studies of β-lactoglobulin interaction with various lipid vesicles. The knowledge of protein-lipid interaction obtained here devotes to the understanding of protein-membrane interaction, including the effect on both protein and lipid vesicles, driving forces and the mechanism for this interaction.

β-lactoglobulin (βLG) is a member of lipocalin protein family and has been shown to be able to interact with various surfactants and lipid vesicles. First, βLG has been shown that while it interacts with phospholipid vesicles, such as DMPG, DOPG and DSPG, it undergoes conformation transition from β-sheets to α-helices. This interaction and orientation with various lipid vesicles at neutral and acidic pH have been studied by spectroscopic techniques. The degree of induced α-helical conformation was dependent of lipid concentration and was affected by the charge of both protein and lipids. Tryptophan fluorescence emission spectra showed that the tertiary structure of protein may be loosened on binding to the vesicle, indicated by increased fluorescence intensity due to the enhanced quantum yield of Trp61 which has been quenched by the adjacent disulfide bond. Fluorescence quenching experiments indicated that Trps may still be shielded from the solvent and that part of the protein is buried into the bilayer of lipid vesicle upon interaction. The orientations of β-sheet strands and α-helices were found to be parallel and perpendicular, respectively, to the lipid bilayer as indicated by polarized FTIR -ATR experiments. Dynamic light scattering (DLS) and Fourier transform infrared (FTIR) were applied to study βLG interaction with DMPG vesicles. It is shown that the interaction expanded the vesicle sizes twice larger but not affected the lipid vesicle transition temperature. Fluorescence studies of trapped calcein demonstrated DMPG induced leakage in vesicles.

Next, to study the mechanism of interaction between βLG and lipid vesicles further, kinetic studies were undertaken using stopped-flow methods. Three lipids, with negatively charged head groups but with different lengths of acyl chain, were used to prepare vesicles. Kinetic CD and fluorescence decays were measured simultaneously to monitor changes in secondary and tertiary structures, respectively. All decays have been fit to either single or double exponential functions. Finally, a three-step model has been proposed to explain the major states seen in the kinetic interaction of βLG and lipid vesicles. The model proposed that the protein looses its tertiary structure upon absorbed onto the vesicle bilayer surface due to electrostatic interaction, refolds to α-helical structure and then inserts into the vesicle bilayer within which the secondary segments are arrange to be at energy minimum.

Third, isothermal titration calorimetry (ITC) has been applied to investigate heat involved in protein-lipid interaction. It indicated that four different thermal contributions were involved in this interaction at and above lipid transition temperature and two thermal contributions below the lipid transition temperature.

Finally, initial aspects of future work related to βLG expression, purification and mutation to obtain mutated sequences are described. The system for expression and purification was developed. The structure of protein was initially measured by CD spectra.