Spectroscopic Studies on Refolding of Reduced and Mutant Lysozymes, and Collagen/Dentin Crosslinking

The reformation of secondary structure for disordered, disulfide reduced hen egg white lysozyme (HEWL) upon interaction with surfactants and lipid vesicles was studied using Circular Dichroism (CD), fluorescence and infrared (IR) spectroscopic techniques. Equilibrium CD studies showed that reduced HEWL when mixed with negatively charged surfactants above critical micelle concentration (CMC), such as sodium dodecyl sulfate (SDS), or with lipid vesicles having negative head groups, such as DMPG at concentration above CMC, can regain helical structure but lose tertiary structure in such environments. Fluorescence studies showed the changes in the local environment of the tryptophan residues. Comparing the results between oxidized HEWL, reduced HEWL and the 0SS and 1SS (with zero and one disulfide bond, respectively) mutants provides an example of the role of disulfide bonds on the protein folding process. With positively charged/zwitterionic surfactants or lipid vesicles, the interactions with reduced HEWL are weaker and thus the degree of helicity recovered is less. Stopped flow dynamics studies showed that both the CD kinetics and the fluorescence kinetics can be fit to two or three exponentials. The faster steps in CD and fluorescence detected kinetics appear to be correlated, which suggests formation of an intermediate on rapid interaction of the surfactant micelles or lipid vesicles and the protein. The slow steps suggest that the protein further rearrange its structure, continue to gain helical content and relax packing of the tryptophan residues, for which the hydrophobic interactions are the major driving forces. ATR-FTIR and fluorescence spectroscopic analysis were used to study the mechanism of interactions of PACs with collagen and with dentin. The PACs used are from vitis vinifera grape seed extract. Two cross-linking agents, glutaraldehyde and carbodiimide hydrochloride, with known cross-linking mechanisms, were selected for comparative analyses. Both the ATR-FTIR and fluorescence results evidence different mechanisms for these three cross-linking agents. A new feature was observed in the ATR-FTIR spectra in PACs-treated collagen and dentin, suggesting a covalent bond formation between collagen and PACs. PACs-treated collagen also shows different patterns of changes of fluorescence from the chemical cross-linkers, further supporting a different mechanism in dentin.