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.
Project description:Surface-induced protein denaturation has important implications for the development of materials that are resistant and/or innocuous to biomolecules. Here, we studied the mechanism of lysozyme (T4L) unfolding on fused silica (FS) using single-molecule methods that provided direct insight into the cause of denaturation. Unfolding of T4L was monitored by Förster resonance energy transfer while simultaneously tracking the adsorption, diffusion, and desorption of individual molecules at the solid-solution interface. Results of high-throughput single-molecule analysis suggested that the unfolding of T4L on FS was mediated by surface diffusion and occurred on isolated nanoscale sites, which were relatively rare and distinct from the majority of the surface. These observations suggest that surface-mediated protein unfolding is a search process that is based on the exploration for denaturing sites by the protein. Ultimately, these findings have important implications for the design of protein-compatible surfaces.
Project description:The enzyme organophosphorous hydrolase (OPH) catalyzes the hydrolysis of a wide variety of organophosphorous compounds with high catalytic efficiency and broad substrate specificity. The immobilization of OPH in functionalized mesoporous silica (FMS) surfaces increases significantly its catalytic specific activity, as compared to the enzyme in solution, with important applications for the detection and decontamination of insecticides and chemical warfare agents. Experimental measurements of immobilization efficiency as a function of the charge and coverage percentage of different functional groups have been interpreted as electrostatic forces being the predominant interactions underlying the adsorption of OPH onto FMS surfaces. Explicit solvent molecular dynamics simulations have been performed for OPH in bulk solution and adsorbed onto two distinct interaction potential models of the FMS functional groups to investigate the relative contributions of nonbonded interactions to the conformational dynamics and adsorption of the protein. Our results support the conclusion that electrostatic interactions are responsible for the binding of OPH to the FMS surface. However, these results also show that van der Waals forces are detrimental for interfacial adhesion. In addition, it is found that OPH adsorption onto the FMS models favors a protein conformation whose active site is fully accessible to the substrate, in contrast to the unconfined protein.
Project description:Experimental evidence suggests that proteins adsorbed to hydrophobic surfaces at low coverages are stabilized relative to the bulk. For larger coverages, proteins unfold and form beta-sheets. We performed computer simulations on model proteins and found that: 1), For weakly adsorbing surfaces, unfolded conformations lose more entropy upon adsorption than folded ones. 2), The melting temperature, both in the bulk and at surfaces, decreases with increasing protein concentration because of favorable interprotein interactions. 3), Proteins in the bulk show large unfolding free energy barriers; this barrier decreases at stronger adsorbing surfaces. We conjecture that typical experimental temperatures appear to be below the bulk melting temperature for a single protein, but above the melting temperature for concentrated protein solutions. Purely thermodynamic factors then explain protein stabilization on adsorption at low concentrations. However, both thermodynamic and kinetic factors are important at higher concentrations. Thus, proteins in the bulk do not denature with increasing concentration due to large kinetic barriers, even though the aggregated state is thermodynamically preferred. However, they readily unfold upon adsorption, with the surface acting as a heterogeneous catalyst. The thermal behavior of proteins adsorbed to hydrophobic surfaces thus appears to follow behavior independent of their chemical specificity.
Project description:We use the statistics of photon emission from single molecules to probe the ultrafast dynamics of an unfolded protein via Förster resonance energy transfer. Global reconfiguration of the chain occurs on a time scale of approximately equal to 50 ns and slows down concomitant with chain collapse under folding conditions. These diffusive dynamics provide a missing link between the phenomenological chemical kinetics commonly used in protein folding and a physical description in terms of quantitative free energy surfaces. The experiments demonstrate the potential of single-molecule methods in accessing the biologically important nanosecond time scales even in heterogeneous populations.
Project description:With the use of single-molecule total internal reflection fluorescence microscopy (TIRFM), the dynamics of bovine serum albumin (BSA) and human fibrinogen (Fg) at low concentrations were observed at the solid-aqueous interface as a function of temperature on hydrophobic trimethylsilane (TMS) and hydrophilic fused silica (FS) surfaces. Multiple dynamic modes and populations were observed and characterized by their surface residence times and squared-displacement distributions (surface diffusion). Characteristic desorption and diffusion rates for each population/mode were generally found to increase with temperature, and apparent activation energies were determined from Arrhenius analyses. The apparent activation energies of desorption and diffusion were typically higher on FS than on TMS surfaces, suggesting that protein desorption and mobility were hindered on hydrophilic surfaces due to favorable protein-surface and solvent-surface interactions. The diffusion of BSA on TMS appeared to be activationless for several populations, whereas diffusion on FS always exhibited an apparent activation energy. All activation energies were small in absolute terms (generally only a few kBT), suggesting that most adsorbed protein molecules are weakly bound and move and desorb readily under ambient conditions.
Project description:In this work, we review the process of protein unfolding characterized by a solid-state nanopore based device. The occupied or excluded volume of a protein molecule in a nanopore depends on the protein's conformation or shape. A folded protein has a larger excluded volume in a nanopore thus it blocks more ionic current flow than its unfolded form and produces a greater current blockage amplitude. The time duration a protein stays in a pore also depends on the protein's folding state. We use Bovine Serum Albumin (BSA) as a model protein to discuss this current blockage amplitude and the time duration associated with the protein unfolding process. BSA molecules were measured in folded, partially unfolded, and completely unfolded conformations in solid-state nanopores. We discuss experimental results, data analysis, and theoretical considerations of BSA protein unfolding measured with silicon nitride nanopores. We show this nanopore method is capable of characterizing a protein's unfolding process at single molecule level. Problems and future studies in characterization of protein unfolding using a solid-state nanopore device will also be discussed.
Project description:Under native conditions, proteins can undergo transient partial unfolding, which may cause proteins to misfold or aggregate. A change in sequence connectivity by circular permutation may affect the energetics of transient partial unfolding in proteins without altering the three-dimensional structures. Using Escherichia coli dihydrofolate reductase (DHFR) as a model system, we investigated how circular permutation affects transient partial unfolding in proteins. We constructed three circular permutants, CP18, CP37, and CP87, with the new N-termini at residue 18, 37, and 87, respectively, and probed transient partial unfolding by native-state proteolysis. The new termini in CP18, CP37, and CP87 are within, near, and distal to the Met20 loop, which is known to be dynamic and also part of the region that undergoes transient unfolding in wild-type DHFR. The stabilities of both native and partially unfolded forms of CP18 are similar to those of wild-type DHFR, suggesting that the influence of introducing new termini in a dynamic region to the protein is minimal. CP37 has a significantly more accessible partially unfolded form than wild-type DHFR, demonstrating that introducing new termini near a dynamic region may promote transient partial unfolding. CP87 has significantly destabilized native and partially unfolded forms, confirming that modification of the folded region in a partially unfolded form destabilizes the partially unfolded form similar to the native form. Our findings provide valuable guidelines to control transient partial unfolding in designing circular permutants in proteins.
Project description:The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.
Project description:The atomistic modeling of protein adsorption on surfaces is hampered by the different time scales of the simulation ([Formula: see text][Formula: see text]s) and experiment (up to hours), and the accordingly different 'final' adsorption conformations. We provide evidence that the method of accelerated molecular dynamics is an efficient tool to obtain equilibrated adsorption states. As a model system we study the adsorption of the protein BMP-2 on graphite in an explicit salt water environment. We demonstrate that due to the considerably improved sampling of conformational space, accelerated molecular dynamics allows to observe the complete unfolding and spreading of the protein on the hydrophobic graphite surface. This result is in agreement with the general finding of protein denaturation upon contact with hydrophobic surfaces.
Project description:We have used the combination of single-molecule Förster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe the conformational ensemble of the collapsed unfolded state of the small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult to access experimentally, because the equilibrium signal in ensemble experiments is dominated by folded molecules. Here, we avoid this problem in two ways. One is the use of single-molecule Förster resonance energy transfer, which allows the separation of folded and unfolded subpopulations at equilibrium and provides information on long-range intramolecular distance distributions. From experiments with donor and acceptor chromophores placed at different positions within the chain, we find that the distance distributions in unfolded CspTm agree surprisingly well with a Gaussian chain not only at high concentrations of denaturant, where the polypeptide chain is expanded, but also at low denaturant concentrations, where the chain is collapsed. The second, complementary approach is synchrotron radiation circular dichroism spectroscopy of collapsed unfolded molecules transiently populated with a microfluidic device that enables rapid mixing. The results indicate a beta-structure content of the collapsed unfolded state of approximately 20% compared with the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, which were previously found only for highly expanded unfolded proteins.