Insights into a Protein-Nanoparticle System by Paramagnetic Perturbation NMR Spectroscopy

Abstract

Background: The interaction between proteins and nanoparticles is a very relevant subject because of the potential applications in medicine and material science in general. Further interest derives from the amyloidogenic character of the considered protein, β2-microglobulin (β2m), which may be regarded as a paradigmatic system for possible therapeutic strategies. Previous evidence showed in fact that gold nanoparticles (AuNPs) are able to inhibit β2m fibril formation in vitro. Methods: NMR (Nuclear Magnetic Resonance) and ESR (Electron Spin Resonance) spectroscopy are employed to characterize the paramagnetic perturbation of the extrinsic nitroxide probe Tempol on β2m in the absence and presence of AuNPs to determine the surface accessibility properties and the occurrence of chemical or conformational exchange, based on measurements conducted under magnetization equilibrium and non-equilibrium conditions. Results: The nitroxide perturbation analysis successfully identifies the protein regions where protein-protein or protein-AuNPs interactions hinder accessibility or/and establish exchange contacts. These information give interesting clues to recognize the fibrillation interface of β2m and hypothesize a mechanism for AuNPs fibrillogenesis inhibition. Conclusions: The presented approach can be advantageously applied to the characterization of the interface in protein-protein and protein-nanoparticles interactions.

Approaching Protein Aggregation and Structural Dynamics by Equilibrium and Nonequilibrium Paramagnetic Perturbation

“…Vast literature documents the use of stable nitroxide free radicals and lanthanide complexes 7,8,10−15 or other paramagnetic transition metal chelates. 16,17 Moving from our earlier work exploring the theoretical foundations and the interpretative insights of the observed evidence, 18,19 we show new valuable applications of extrinsic spin labels in protein NMR that complement the classical identification of exposed regions. 20,21 We name the presented approach PENELOP, that is, Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation.…”

“…Different relaxation delays were used to acquire the data, 0.5 and 4 s for D76N β2m and 0.3 and 3 s for Nsp9, according to the criteria reported earlier. 18,19 All NMR data were processed with TOPSPIN version 4.0.2. Prior to Fourier transformation, linear prediction in t1 (up to 192 points) and zero filling were applied to yield the final data set of 2 K × 1 K points.…”

“…Compared to the relative intensity (RI) values commonly employed in PRE studies, that is, those obtained from the ratios of the individual signal intensities in the presence and absence of paramagnetic probes, the adopted normalized attenuation definition conveys, at a glance, the additional information concerning the perturbation extent with respect to the average effects. From the previous definitions, the error on the individual A N values can be calculated as: 19…”

Protein Adsorption and Conformational Changes

“…Moreover, nanoparticle surface curvature has a profound influence on the adsorption process. Thus, protein-nanoparticle interactions have been extensively investigated, and it is not surprising that they are discussed in a number of reports in this Special Issue [ 12 , 13 , 14 , 15 ]. Lipid surfaces are instead the subject of another contribution in this issue [ 16 ].…”

“…Protein interaction with gold nanoparticles is also the subject discussed in another original report of this issue, contributed by Hunashal et al [ 13 ], although in this case, the focus is on the influence of nanoparticles on protein fibrillogenesis. The authors introduce a method of investigation of site-specific transient interactions based on NMR signal perturbations induced by an extrinsic paramagnetic agent.…”