Small noble metal nanoclusters can be formed in situ by direct reduction and stabilization of a metal precursor by biomolecules such as proteins. Considering the diversity in amino acid composition of proteins, and hence their reductive ability, a general method for synthesis of gold nanoclusters using proteins is presented here. A range of proteins (bovine serum albumin, fibrinogen, a-lactalbumin, lysozyme, cytochrome c, myoglobin, b-lactoglobulin and a-chymotrypsin) have been studied, based on size, isoelectric point, flexibility and 3-dimensional structure. Results show protein-gold nanoconstructs with complex protein-specific photophysical properties. The effect on the 3-dimensional conformation of the proteins upon formation of gold nanoclusters and/or nanoparticles within the protein structure is also shown to be highly protein-dependent. A general mechanism for the formation of protein-gold nanoconstructs is proposed, based on charge density matching, yielding a high local concentration of the metal precursor on the protein structure which in turn can nucleate, grow and be stabilized by amino acid residues in the protein.

By adsorbing bovine serum albumin (BSA) on gold nanoparticles (Aunps) with diameters 30 nm and 80 nm, different degrees of protein unfolding were obtained. Adsorption and adlayer conformation were characterized by UV-vis spectroscopy, ζ-potential measurements, steady-state and time-resolved fluorescence. The unfolding was also studied using 1-anilino-8-naphthalene sulfonate (ANS) as an extrinsic probe, showing that BSA unfolds more on 80 nm Aunp than on 30 nm Aunp. Langmuir monolayer studies using two distinct methods of introducing the BSA and BSA-Aunp constructs accompanied with Brewster Angle Microscopy (BAM) and Digital Video Microscope (DVM) imaging demonstrated that BSA-Aunp constructs induce film miscibility with L-α-phosphatidylethanolamine not seen for BSA or Aunp alone. The changes induced by partial unfolding clearly give better film-penetration ability, as well as disruption of liquid crystalline domains in the film, thereby inducing film miscibility. Gold or protein only does not possess the nanoscale film-affecting properties of the protein-gold constructs, and as such the surface-active and miscibility-affecting characteristics of the BSA-Aunp represent emergent qualities.

Protein-stabilized gold nanoconstructs are widely studied due to their potential applications in biosensing, drug and gene delivery, and bioimaging. While a number of studies have focused on the novel properties of such materials emanating from the gold, there has been little focus on how the protein shell is affected by nanocluster formation with respect to conformation, stability and function. Herein, we show the synthesis of protein-stabilized gold nanoconstructs varying in size from small clusters (~8 Au atoms) dispersed within proteins to nanoparticles stabilized by multiple proteins by varying the concentration of gold precursor and reducing agent. Proteins used were bovine serum albumin (BSA), bovine a-lactalbumin (BLA) and lysozyme (LYZ). Photophysical properties of the gold nanostructures were monitored using UVvis and fluorescence measurements, revealing that the gold constructs can be tuned from luminescent clusters to nanoparticles displaying localized surface plasmon resonance (LSPR). Conformational changes of the protein following conjugation to gold nanostructures were studied using steady-state and timeresolved Trp fluorescence measurements and circular dichroism. The degree of conformational perturbation varied greatly between the proteins used, with BLA being the most tunable in terms of gradual unfolding, whereas the conformational stability of LYZ was very sensitive to the reducing agent used. To assess the impact of the gold nanostructures as well as the reducing agent on protein function, the LYZ-gold nanoconstructs were subjected to an activity test by degradation of Micrococcus lysodeikticus cell walls, revealing that the activity of the LYZ-Au constructs was retained and tunable, albeit at attenuated levels.

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Human α-Lactalbumin made lethal to tumor cells (HAMLET), and its bovine analogue BAMLET, have in the recent years shown promising results in cancer therapy. HAMLET contains several oleic acids which in turn stabilize a partially unfolded conformational state of the protein, inducing a higher surface activity compared to the native protein, and likely play a role in its cancer-detrimental function as well. Herein, we report the formation of gold-bovine α-lactalbumin nanoconstructs (Au–LA NCs) displaying conformational changes in the native protein and its concomitant ability to reduce HeLa cell viability approaching that of BAMLET. Modification of LA with gold was achieved via two synthesis protocols; (i) utilizing the intrinsic reduction potential of LA (Au–LAint) or (ii) the addition of an extrinsic reducing agent (Au–LAext). The gold–protein nanostructures formed were investigated using a palette of analytical probes including AFM, TEM, ζ-potential, UV–vis, circular dichroism, and fluorescence (steady-state and time-resolved). Toxicity toward HeLa cells was studied using a trypan blue assay and benchmarked against BAMLET. Whereas constructs from both synthetic protocols employed result in conformational changes of the protein and altered surface activity compared to the native protein, Au–LAint was found to display lipid-specific interaction and severe disruption of lipid monolayers. Au–LAint also revealed toxicity toward HeLa-cells comparable to that of BAMLET. The results imply that formation of Au–LA nanoconstructs could be a new route to making HAMLET-like materials, while also imparting a built-in optical probe from fluorescent or plasmonic gold species.