Capturing Conformation-Dependent Molecule–Surface Interactions When Surface Chemistry Is Heterogeneous

Molecular building blocks, such as carbon nanotubes and DNA origami, can be fully integrated into electronic and optical devices if they can be assembled on solid surfaces using biomolecular interactions. However, the conformation and functionality of biomolecules depend strongly on the local chemical environment, which is highly heterogeneous near a surface. To help realize the potential of biomolecular self-assembly, we introduce here a technique to spatially map molecular conformations and adsorption, based on single-molecule fluorescence microscopy. On a deliberately patterned surface, with regions of varying hydrophobicity, we characterized the conformations of adsorbed helicogenic alanine-lysine copeptides using Förster resonance energy transfer. The peptides adopted helical conformations on hydrophilic regions of the surface more often than on hydrophobic regions, consistent with previous ensemble-averaged observations of α-helix surface stability. Interestingly, this dependence on surface chemistry was not due to surface-induced unfolding, as the apparent folding and unfolding dynamics were usually much slower than desorption. The most significant effect of surface chemistry was on the adsorption rate of molecules as a function of their initial conformational state. In particular, regions with higher adsorption rates attracted more molecules in compact, disordered coil states, and this difference in adsorption rates dominated the average conformation of the ensemble. The correlation between adsorption rate and average conformation was also observed on nominally uniform surfaces. Spatial variations in the functional state of adsorbed molecules would strongly affect the success rates of surface-based molecular assembly and can be fully understood using the approach developed in this work

Surface Chemistry Influences Interfacial Fibrinogen Self-Association

Surface chemistry modifications have been exploited in many applications in order to tune protein adsorption, layer formation, and aggregation. However, the kinetic processes by which surface chemistry influences protein adsorption and aggregation remain elusive. By combining intermolecular resonance energy transfer (RET) with high-throughput single-molecule tracking, we compared the dynamics of fibrinogen (Fg) interfacial self-associations on surfaces modified with hydrophobic trimethyl silane (TMS) or hydrophilic oligoethylene glycol (OEG). We directly observed interfacial, dynamic, and reversible Fg–Fg associations from low-RET (unassociated) to high-RET (associated) states. While isolated Fg molecule–TMS surface interactions were weaker than isolated Fg–OEG interactions, increasing protein concentration resulted in a more dramatic decrease in desorption from TMS than from OEG, such that at higher concentrations, Fg desorbed from TMS more slowly than from OEG. In addition to this observation, unassociated molecules were more likely to associate on TMS than on OEG, suggesting that the TMS surface promoted protein–protein associations. Importantly, increasing protein concentration also resulted in a greater increase in the length of time proteins remained associated (i.e., contact times) on TMS than on OEG, such that contact times were longer on TMS than on OEG at higher concentrations but shorter at low concentration, mirroring the behavior of the overall surface residence times. These findings strongly suggest that surface chemistry not only influences protein–surface interactions but can also promote interfacial aggregation on one surface (hydrophobic TMS) relative to another (hydrophilic OEG), and that the latter may well be the more important factor at higher surface coverage

Interfacial Protein–Protein Associations

While traditional models of protein adsorption focus primarily on direct protein–surface interactions, recent findings suggest that protein–protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein–protein associations of bovine serum albumin on polyethylene glycol modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface – with areas of high protein density (i.e., strongly interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e., partial-RET (monomer–monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e., clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage

Dense Poly(ethylene glycol) Brushes Reduce Adsorption and Stabilize the Unfolded Conformation of Fibronectin

Polymer brushes, in which polymers are end-tethered densely to a grafting surface, are commonly proposed for use as stealth coatings for various biomaterials. However, although their use has received considerable attention, a mechanistic understanding of the impact of brush properties on protein adsorption and unfolding remains elusive. We investigated the effect of the grafting density of poly­(ethylene glycol) (PEG) brushes on the interactions of the brush with fibronectin (FN) using high-throughput single-molecule tracking methods, which directly measure protein adsorption and unfolding within the brush. We observed that, as grafting density increased, the rate of FN adsorption decreased; however, surface-adsorbed FN unfolded more readily, and unfolded molecules were retained on the surface for longer residence times relative to those of folded molecules. These results, which are critical for the rational design of PEG brushes, suggest that there is a critical balance between protein adsorption and conformation that underlies the utility of such brushes in physiological environments

Connecting Protein Conformation and Dynamics with Ligand–Receptor Binding Using Three-Color Förster Resonance Energy Transfer Tracking

Specific binding between biomolecules, i.e., molecular recognition, controls virtually all biological processes including the interactions between cells and biointerfaces, both natural and synthetic. Such binding often relies on the conformation of biomacromolecules, which can be highly heterogeneous and sensitive to environmental perturbations, and therefore difficult to characterize and control. An approach is demonstrated here that directly connects the binding kinetics and stability of the protein receptor integrin α_vβ₃ to the conformation of the ligand fibronectin (FN), which are believed to control cellular mechanosensing. Specifically, we investigated the influence of surface-adsorbed FN structure and dynamics on α_vβ₃ binding using high-throughput single-molecule three-color Förster resonance energy transfer (FRET) tracking methods. By controlling FN structure and dynamics through tuning surface chemistry, we found that as the conformational and translational dynamics of FN increased, the rate of binding, particularly to folded FN, and stability of the bound FN−α_vβ₃ complex decreased significantly. These findings highlight the importance of the conformational plasticity and accessibility of the arginine-glycine-aspartic acid (RGD) binding site in FN, which, in turn, mediates cell signaling in physiological and synthetic environments