Lysozyme encapsulated gold nanoclusters: effects of cluster synthesis on natural protein characteristics

The study of gold nanoclusters (AuNCs) has seen much interest in recent history due to their unique fluorescence properties and environmentally friendly synthesis method using proteins as a growth scaffold. The differences in the physicochemical properties of lysozyme encapsulated AuNCs in comparison to natural lysozyme are characterised in order to determine the effects AuNCs have on natural protein behaviour. The hydrodynamic radius (dynamic light scattering), light absorbance (UV-Vis), electrophoretic mobility, relative density, dynamic viscosity, adsorption (quartz crystal microbalance) and circular dichroism (CD) characteristics of the molecules were studied. It was found that lysozyme forms small dimer/trimer aggregates upon the synthesis of AuNCs within the protein. The diameter of Ly-AuNCs was found to be 8.0 nm across a pH range of 2-11 indicating dimer formation, but larger aggregates with diameters >20 nm were formed between pH 3 and 6. The formation of larger aggregates limits the use of Ly-AuNCs as a fluorescent probe in this pH range. A large shift in the protein's isoelectric point was also observed, shifting from 11.0 to 4.0 upon AuNC synthesis. This resulted in major changes to the adsorption characteristics of lysozyme, observed using a QCM. A monolayer of 8 nm was seen for Ly-AuNCs at pH 4, offering further evidence that the proteins form small aggregates, unlike the natural monomer form of lysozyme. The adsorption of Ly-AuNCs was seen to decrease as pH was increased; this is in major contrast to the lysozyme adsorption behaviour. A decrease in the α-helix content was observed from 25% in natural lysozyme to 1% in Ly-AuNCs. This coincided with an increase in the β-sheet content after AuNC synthesis indicating that the natural structure of lysozyme was lost. The formation of protein dimers, the change in the protein surface charge from positive to negative, and secondary structure alteration caused by the AuNC synthesis must be considered before attempting to utilise Ly-AuNCs as in vivo probes.