Exploring the Residue-Level Interactions between the R2ab Protein and Polystyrene Nanoparticles

Abstract

In biological systems, proteins can bind to nanoparticles to form a "corona" of adsorbed molecules. The nanoparticle corona is of high interest because it impacts the organism's response to the nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. Ultimately, a residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Through lysine methylation, we observe subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX measurements reveal that certain regions of the R2ab protein undergo faster exchange rates in the presence of PSNPs, suggesting conformational changes upon binding. Both results support a recently proposed "adsorbotope" model, wherein adsorbed proteins consist of unfolded anchor points interspersed with regions of partial structure. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how proteins respond to nanoparticle surfaces, this research emphasizes the need for advanced methods to comprehend these intricate interactions fully at the residue level.

Keywords: adsorbotope; corona; interaction; nanoparticle; protein; structure.

Figure 1.

Peptide fragments of R2ab and the lysines in the respective fragments that are detected by mass spectrometry are represented by unique colors for identification.

Figure 2.

Degrees of methylation show only subtle changes regardless of the presence of PSNPs (A). Error bars represent the standard deviation from three separate experiments. For comparison, regions of R2ab have been colored to show how well the individual peptide fragments have been methylated in the native form (B), in the presence of 50 nm (C), 100 nm (D) and 200 nm (E) PSNPs. Regions which are change from being more methylated to less methylated, (red to blue) indicate possible changes in protein orientation and disorder.

Figure 3.

HDX kinetics of the residues 701 (A), 785 (B), and 844 (C) in the absence (top) and presence (bottom) of PSNPs. Red points represent the measured intensity of each 2D NMR peak for each assigned residue after normalization of the initially collected SOFAST-HMQC time point. The black line represents the fit of the exponential decay as described in the text.

Figure 4.

(A) HDX rates (k_ex) for R2ab where rates could be compared with (red) and without (black) PSNPs. (B) Ratios of k_ex values for each residue; larger rates represent faster exchange in the presence of PSNPs. A black dashed line represents the average rate. (C) Correlation between rates taken from (A). The black dashed line represents the equation y = x. The dotted line represents the best fit of rates through the origin. (D) A comparison of rates mapped onto the structure of R2ab. Detectable residues are shown as CPK spheres. Residues with faster HDX rates relative to the average k_ex ratio are represented on the R2ab structure in red, while those with slower rates are shown in blue. Neutral residues, similar to the average shown in (B), are plotted in white.