Title
Reduced and mutant lysozyme refolding with lipid vesicles. Model study of disulfide impact on equilibria and dynamics
Author
Weying Zhu, Robert Silvers, Harald Schwalbe, Timothy A. Keiderling
Year
2016
Journal
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Abstract
The recovery of secondary structure in disordered, disulfide-reduced hen egg white lysozyme (HEWL) upon interaction with lipid vesicles was studied using circular dichroism (CD), fluorescence and infrared (IR) spectroscopic techniques. Lipid vesicles having negative head groups, such as DMPG, interact with reduced HEWL to induce formation of more helical structure than in native HEWL, but no stable tertiary structure was evident. Changes in tertiary structure, as evidenced by local environment of the tryptophan residues, were monitored by fluorescence. Spectra for oxidized HEWL, reduced HEWL and mutants with no or just one disulfide bond developed variable degrees of added helicity when added to negatively charged lipid vesicles, mostly depending on packing of tails. When mixed with zwitterionic lipid vesicles, reduced HEWL developed β-sheet structure with no increase in helicity, indicating an altered interaction mechanism. Stopped flow CD and fluorescence dynamics, were fit to multi-exponential forms, consistent with refolding to metastable intermediates of increasing helicity for HEWL interacting with lipid vesicles. Formation of an intermediate after rapid interaction of the lipid vesicles and the protein is supported by the correlation of faster steps in CD and fluorescence kinetics, and appears driven by electrostatic interaction to large part. In subsequent slower steps, the partially refolded intermediate further alters structure, gaining helicity and modifying tryptophan packing, as driven by hydrophobic interactions.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Tertiary structure, Thermal stability, Vesicle interactions, Protein folding, Stopped-flow, Kinetics, Biochemistry