Probing the Effects of Cysteine Residues on Protein Adsorption onto Gold Nanoparticles Using Wild-Type and Mutated GB3 Proteins

Abstract

The role of cysteine residues in the protein binding kinetics and stability on gold nanoparticles (AuNP) was studied using AuNP localized surface plasmon resonance (LSPR) in combination with an organothiol (OT) displacement method. GB3, the third IgG-binding domain of protein G, was used to model protein-AuNP adsorption. While wild-type GB3 (GB30) contains no cysteine residues, bioengineered GB3 variants containing one (GB31) and two (GB32) cysteine residues were also tested. The cysteine content has no significant effect on GB3 binding kinetics with AuNPs, and most protein adsorption occurs within the first few seconds upon protein/AuNP mixing. However, the stability of GB3 on the AuNP surface against OT displacement depends strongly on the cysteine content and the age of the AuNP/GB3 mixture. The GB30 covered AuNPs can be completely destabilized and aggregated by OTs, regardless of the age of the GB30/AuNP mixtures. Long-time incubation of GB31 or GB32 with AuNPs can stabilize AuNPs against the OT adsorption inducted aggregation. This study indicates that multiple forces involved in the GB3/AuNP interaction, and covalent binding between cysteine and AuNP is essential for a stable protein/AuNP complex.

Gold Nanoparticle-Based Facile Detection of Human Serum Albumin and Its Application as an INHIBIT Logic Gate

“…It has been reported that the cysteine residue is critical in maintaining the stability of Au NPs against aggregation. 39,40 Accordingly, the fact that amino acids, peptides and proteins investigated here failed to protect the Au NPs against the melamine-induced aggregation is supposed to be due to the lack of free cysteine residues exposed on the surfaces (except cysteine and glutathione). In addition, although cysteine and glutathione bear free cysteine groups that allow their binding to the surface of Au NPs through the formation of Au-S bonds, they present almost no anti-aggregation effect.…”

“…Previous studies demonstrated that amino acids and peptides are usually adsorbed on the surface of Au NPs through their amino groups and thiol groups (for cysteine and glutathione). [38][39][40] Here, the selectivity was evaluated by testing the response of the assay to a with Au NPs through their amino acid residues. Thus, selectivity of the colorimetric method toward these proteins was also investigated.…”

Theranostic potential of gold nanoparticle-protein agglomerates

“…13,40 The first kind of possible interaction involves the electrostatic force of attraction, whereby the positively charged amino acids (such as arginine and lysine 36 ) among the surface-exposed residues of a protein molecule can form bonds with negatively charged citrate groups present at the surface of the Au NP at physiological pH. 13,21,40 In this situation, the protein molecules do not interact directly with the Au NP; instead the citrates (stabilizing agent) essentially act as an interface between the attached protein and the surface of Au NP. 13 In the second possibility, the protein molecules can directly get adsorbed onto the Au NP surface by displacing the citrate moieties.…”

“…25,41,42 It is believed that, the formation of S-Au bond(s) likely occur(s) after the initial binding of the protein with the Au NPs through forces such as electrostatic interaction and van der Waals forces. 40 It has been shown that BSA binds to the Au NPs more strongly than organothiols (including glutathione, cysteine and mercaptobenzimidazole) due to formation of several S-Au bonds through its 35 cysteine residues. 43 Clearly, the number of cysteine residues and their availability (whether surface-exposed or buried into protein interior) at the surface of the Au NPs in a protein are expected to determine the degree of the cysteine -Au NP interaction and the number of resulting S-Au bonds.…”

“…26,37,47,48 The formation of Au-S covalent bonds through the surface exposed cysteine residues of α-amylase is tenable to culminate in the partial unfolding of the protein over the surface of Au NP. 26,37,40 The adsorbed layer of unfolded α−amylase around the Au NPs -in a fashion much similar to lysozyme as described above -can act as 'hot-spots' for further protein-protein interaction with unbound The adsorption of protein onto the surface of Au NP can involve (i) the electrostatic attraction (positively charged amino acids and negatively charged citrates at the Au NP surface) or (ii) direct interaction of the protein with the Au NP by displacing the citrates, or (iii) combination of both these processes. (iv) The protein molecules directly interacting with the Au NP 'unfold' over the NP surface exposing hydrophobic patches, which helps unfolding other protein molecules interacting with the Au NPs.…”