Exploring Residue-Level Interactions between the Biofilm-Driving 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 significant interest because it impacts an organism's response to a nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. 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. Lysine methylation experiments reveal subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX rates become slower overall in the presence of PSNPs. However, some regions of the R2ab protein exhibit faster than average exchange rates in the presence of PSNPs, while others are slower than the average behavior, suggesting conformational changes upon binding. HDX rates and methylation ratios support a recently proposed "adsorbotope" model for PSNPs, wherein adsorbed proteins consist of unfolded anchor points interspersed with partially structured regions. 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 R2ab adsorbs onto PSNP surfaces, this research emphasizes the need for advanced methods to comprehend residue-level interactions in the nanoparticle corona.

Figure 1.

Peptide fragments of R2ab and the lysine residues in the respective fragments detected by mass spectrometry are shaded uniquely for identification. Gray regions represent those peptides that were not detected (residues 691–698, 745–750, 846–847) and detected peptides not containing a lysine.

Figure 2.

Degree of methylation in R2ab-PSNP mixtures. (A) Degree of methylation of R2ab in the absence and presence of PSNPs of varying sizes, quantified by LC and identified by MS/MS. The bars are colored corresponding to R2ab without any PSNPs (blue), with 50 nm PSNPs (red), with 100 nm PSNPs (green), and with 200 nm PSNPs (violet). (B–D) Degree of methylation relative to no PSNPs, mapped onto the R2ab structure in the presence of (B) 50 nm PSNPs, (C) 100 nm PSNPs, and (D) 200 nm PSNPs. Fragments from panel A are colored over the gradient range shown. Fragments with no lysine residues are colored black. The left- and right-hand columns are rotated 180° with respect to the axis shown.

Figure 3.

HDX kinetics of the residues (A) 701, (B) 785, and (C) 844 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 ($k_{\text{ex}}$) for R2ab, where rates could be compared with (red) and without (black) PSNPs. (B) Ratios of $k_{\text{ex}}$ values for each residue; larger ratios 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 $k_{\text{ex}}$ 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.