Spectroscopic Studies of Model Protein Interactions with Lipid Vesicles and Insulin Fibril Disassembly
thesisposted on 22.10.2017, 00:00 by Ge Zhang
The work in this thesis focuses on spectroscopic studies of refolding and interaction of model proteins with various lipid vesicles and on dimethyl sulfoxide (DMSO)-induced disassembly of various structural variants of insulin fibrils. The main spectroscopic methods utilized in this thesis include electronic circular dichroism (ECD), fluorescence, infrared absorption (IR) and vibrational circular dichroism (VCD). ECD, IR and VCD were used to characterize the secondary structure of model proteins based on their distinct bandshapes for various secondary structure elements. Polarized attenuated total reflectance (ATR)-FTIR was particularly useful in a protein-lipid membrane study (Chapter 3), because it can provide information about relative orientation of protein segments and the lipid bilayer. VCD was used to probe the disassembly of insulin fibrils (Chapter 4) due to its sensitivity to supramolecular chirality arising from higher-order self-assembly of aggregates. Fluorescence was used to monitor protein local tertiary structural change in protein-lipid vesicle interaction. In Chapter 3, a β-sheet to α-helix transformation of monomeric β-lactoglobulin (βLG) induced by small unilamellar vesicles (SUVs) of zwitterionic lipids at low pH was determined via various spectroscopic techniques. With SUVs of a zwitterionic lipid (1, 2-distearoyl-sn-glycero-3-phosphocholine, DSPC), βLG converted to a substantially helical form in a two-step kinetic process (fast and slow steps) monitored by CD. Fluorescence implied a rapid initial change in the Trp environments followed by a slower process paralleling the secondary structure change. Polarization ATR-FTIR results indicate the helices formed are at least partially inserted into the lipid bilayer and the sheet segments are on the surface. Thermal behavior showed that changes in the secondary structure for the lipid bound βLG occurred in three phases: the first is a slight reduction of the α-helix for βLG in the protein-lipid complex; the second is the DSPC phase change after which the proteins apparently dissociated from the vesicles and refolded into their native structures; the third is the unfolding of solvated βLG at high temperature. These thermal and kinetic behaviors suggest a different mechanism for the monomeric βLG interaction with zwitterionic lipids than was seen previously for the dimeric form at higher pH. In Chapter 4, VCD was utilized to characterize the macroscopic chirality and the DMSO-induced disassembly process for two types of insulin fibrils formed under different conditions. In this study it is confirmed that very high concentrations of DMSO both disaggregate these insulin fibrils and change their secondary structure. Inter-conversion of some insulin fibril types also occurred during the destabilization process as monitored by VCD. Transmission electron microscopy (TEM) images correlated the change in VCD sign pattern to alteration of morphology of the insulin fibrils. In Chapter 5, a computationally designed outer membrane protein (OmpFG) expected to form a monomeric β-barrel in the membrane was expressed and studied. At least partial refolding was evidenced in the lipid vesicles by CD detection of secondary structural change (random coil to β-sheet) and the change to a less polar environment of Trp residues was monitored by fluorescence. The results of a dye leakage assay indicate that the OmpFG can interact with lipid vesicles and may form a pore-like structure with relatively high conductivity.