Reduced and mutant lysozyme refolding with lipid vesicles. Model study of disulfide impact on equilibria and dynamics

• Reduced and Cys → Ala mutated lysozymes refold to more than native helicity in lipids.
• Refolded helical segments mostly insert in lipid bilayer, sheet parts on surface.
• Native lysozyme has only small secondary structure change with lipid vesicles.
• Reduced lysozyme refolding kinetics in lipid vesicles are faster than renaturation.
• Kinetics indicate that lipid interaction mechanism differs from that with surfactants.

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 increased 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 change 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 largely appears driven by electrostatic interaction. In subsequent slower steps, the partially refolded intermediate further alters structure, gaining helicity and modifying tryptophan packing, as driven by hydrophobic interactions.

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