Quantitative Measurement of Multiprotein Nanoparticle Interactions Using NMR Spectroscopy

Abstract

An effective intensity-based reference is a cornerstone for quantitative nuclear magnetic resonance (NMR) studies, as the molecular concentration is encoded in its signal. In theory, NMR is well suited for the measurement of competitive protein adsorption onto nanoparticle (NP) surfaces, but current referencing systems are not optimized for multidimensional experiments. Presented herein is a simple and novel referencing system using 15 N tryptophan (Trp) as an external reference for 1 H− 15 N 2D NMR experiments. The referencing system is validated by the determination of the binding capacity of a single protein onto gold NPs. Then, the Trp reference is applied to protein mixtures, and signals from each protein are accurately quantified. All results are consistent with previous studies, but with substantially higher precision, indicating that the Trp reference can accurately calibrate the residue peak intensities and reduce systematic errors. Finally, the proposed Trp reference is used to kinetically monitor in situ and in real time the competitive adsorption of different proteins. As a challenging test case, we successfully apply our approach to a mixture of protein variants differing by only a single residue. Our results show that the binding of one protein will affect the binding of the other, leading to an altered NP corona composition. This work therefore highlights the importance of studying protein−NP interactions in protein mixtures in situ, and the referencing system developed here enables the quantification of binding kinetics and thermodynamics of multiple proteins using various 1 H− 15 N 2D NMR techniques.

Understanding the Adsorption of Peptides and Proteins onto PEGylated Gold Nanoparticles

“…The adsorption of the selected proteins is tight and in slow exchange timescale with no line broadening, rendering their NMR signals invisible upon attachment to the AuNP surface. Therefore, the remaining protein NMR signals correspond to the unbound protein amount [ 38 , 50 ]. Examples of quantifying protein unbound concentrations using 1D NMR for BSA and 2D NMR for K19C GB3 are presented in Figures S3 and S4, Supporting Information .…”

“…For comparison, the binding of these biomolecules onto non-PEGylated AuNPs is extremely fast and results in higher binding capacities. Previous studies show that ~90% of protein binding to citrate-coated AuNPs occurs in the initial ~5 min, and adsorption is completed within an hour [ 38 , 39 ]. The binding capacities (molecules per AuNp) for GSH, WT GB3 and BSA on bare AuNPs were determined to be , , and , respectively [ 37 ].…”

“…Here, we design a simple test that examines how protein size, charge, and surface cysteine content influences adsorption for several different PEGylation densities on AuNPs, holding the size of the core AuNP constant. Our approach employs a newly developed external NMR referencing method for quantifying protein binding to AuNPs in situ, and our observations do not require displacement of proteins or treatment of the AuNPs [ 38 ]. This approach enables straightforward measurements of adsorption kinetics, provided no line broadening is observed in the biomolecular NMR spectra.…”

“…Protein samples were mixed with AuNPs at concentration of 120 nM AuNPs. Quantitation was performed using an external standard, described previously [ 38 ]. This approach is effective when adsorption is slow on the NMR timescale, and when no line broadening is observed, as occurs here [ 38 , 50 ].…”

“…The AuNPs are coated with 5K, 10K, and 30K thiolated PEG, allowing us to probe different levels of PEG coverage. Using NMR spectroscopy [ 37 , 38 , 39 ], we have quantified the kinetics of and final stoichiometry of biomolecular adsorption to PEGylated AuNPs, and we discuss trends and physical properties that lead to efficient binding to PEGylated AuNP surfaces.…”