Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

Abstract

A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.protein adhesion | single-molecule fluorescence | total internal reflection fluorescence microscopy U nderstanding the effect of near-surface environments on protein conformation is critical in many bioengineering and biomedical applications, including biosensing, cell culture, tissue engineering, biocatalysis, and pharmaceutical formulation. Importantly, surface interactions that perturb protein structure can inactivate proteins, as has widely been observed in the case of surface-immobilized enzymes (1-6). Such interactions, by inducing unfolding and subsequent accumulation of freely absorbing proteins on biomaterial surfaces, may trigger unfavorable cellular responses (7-10). However, experimental methods to elucidate both protein structure and interfacial dynamics (e.g., adsorption, diffusion, desorption), particularly in heterogeneous near-surface environments, are virtually nonexistent.Conventional methods to determine surface effects on protein structure are largely ensemble-averaging techniques that provide limited mechanistic insight. Such limitations can lead to misinterpretations of apparent interfacial phenomena, which are used to explain the biocompatibility of biomaterials. For example, conventional biophysical methods often find that the average surface protein conformation relaxes from ...

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

“…55 Thus, the initial conformation (measured immediately after adsorption) favored the folded state when compared to the final conformation (measured in the frame prior to desorption). We performed a similar analysis here, comparing the likelihood of observing the helix state immediately after adsorption and immediately prior to desorption ( Figure 5A).…”

Interfacial Protein–Protein Associations

“…Due to the large spectral separation of Alexa Fluor 555 and Alexa Fluor 647, no significant direct excitation of acceptor or donor bleed-through into the acceptor channel was observed in our experiments, as was demonstrated previously for this optical system and RET-pair. 37 Channels were aligned to within 1-2 pixels with an alignment grid prior to experiments. Fine sub-pixel alignment corrections were made before object identification and tracking analysis by convolving the two channels as a function of an offset and identifying the offset that maximized the intensity-intensity product of the two channels, as described previously.…”

A bottom-up approach to understanding protein layer formation at solid–liquid interfaces

“…Parameters to describe the kinetic behavior and relative prevalence of these states can be taken directly from available SMT literature. While it may be possible to directly observe different conformations [175] or aggregation states [176] at the single-molecule level, from a practical perspective, these states can be characterized by their dynamic behaviors (e.g. different desorption rates or diffusion coefficients) without fully understanding the molecular structure of each state.…”

“…Such an approach was recently demonstrated in which site-specific fluorescence labeling allowed a conformational change in freely-adsorbing organophosphorous hydrolase to be measured at the single-molecule level. [175] …”