A three-step model for protein-gold nanoparticle adsorption

Authors: ZHANG, DONGMAO - Mississippi State University; WANG, AILIN - Mississippi State University; VANGALA, KARTHIKESHWAR - Mississippi State University; VO, TAM - Mississippi State University; FITZKEE, NICHOLAS - Mississippi State University

Submitted to: Journal of Physical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/21/2014
Publication Date: 3/21/2014
Citation: Zhang, D., Wang, A., Vangala, K., Vo, T., Fitzkee, N.C. 2014. A three-step model for protein-gold nanoparticle adsorption. Journal of Physical Chemistry. 118:8134-8142.

Interpretive Summary: We have presented an NMR-based approach for characterizing the stoichiometry of molecular binding to AuNPs. The approach is straightforward and rapid, and under saturating conditions, it is possible to estimate the maximum adsorption capacity. We measured the stoichiometry for a data set of six proteins, and we found that the adsorption capacity can be predicted using the radius of gyration calculated from the folded protein structure. Our data support a simple model for protein adsorption on 15 nm AuNPs: Initially, protein binding is reversible, and the structural changes upon adsorption are minor, allowing the protein to remain globular. Once on the surface, the protein is able to reorient, making room for neighboring proteins so that the adsorption capacity is maximized. Finally, cysteine-containing proteins experience a hardening step where binding becomes irreversible. This final step is likely associated with surface restructuring that would lead to changes in the SPR and SERS signal from the bound protein. The methodology outlined here can be easily applied to other bio-conjugate systems with tight binding, and future work will investigate the behavior of proteins on other nanoparticle systems that would provide the foundation for the development of sensitive spectroscopic detection of food pathogens using gold nanoparticles.

Technical Abstract: Gold nanoparticles (AuNPs) are an attractive delivery vector in biomedicine because of their low toxicity and unique electronic and chemical properties. AuNP bioconjugates can be used in many applications, including nanomaterials, biosensing, and drug delivery. While the phenomenon of spontaneous protein–AuNP adsorption is well-known, the structural and mechanistic details of this interaction remain poorly understood. As a result, predicting the orientation and structure of proteins on the nanoparticle surface remains a challenge. New techniques are therefore needed to characterize the structural properties of proteins as they bind to AuNPs. We have developed a straightforward and rapid NMR-based approach to quantitatively characterize the protein–AuNP interaction. This approach is immune to the inner filter effect, which complicates fluorescence measurements, and it can be performed without prior centrifugation of samples. Using a data set of six proteins, ranging in size from 3 to 583 residues, we measured the stoichiometry of binding to AuNPs with a diameter of 15 nm. The stoichiometry of binding can be predicted based on simple geometric considerations assuming that proteins remain globular on the AuNP surface. Using our approach, we find that a protein lacking cysteine residues can be displaced from AuNPs using a small organothiol compound, but proteins with surface cysteines are resistant to displacement. From this data we develop a model for adsorption consisting of three steps: an initial reversible association step, a rearrangement/reorientation step on the AuNP surface, and a final cysteine-dependent “hardening” step, after which binding becomes irreversible.