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Transition paths are fleeting events when a molecule crosses a barrier separating stable configurational basins. Recent advances in single molecule experiments, including optical tweezers-based force spectroscopy and FRET-based techniques, and data analysis methods have allowed detection of various statistical properties of transition paths. These observations have highlighted an important limitation in their current theoretical interpretation – a model of diffusive dynamics of the reaction coordinate along a one-dimensional free energy landscape is often unable to account for measurements of transition path time distributions in single molecule experiments. Here, we report similar observations in a long all-atom simulation of a small fast folding protein that exhibits multiple folding and unfolding transitions. Specifically, we discovered that the distribution of transition path times in this case is much broader than the prediction of the one-dimensional diffusion model. Moreover, direct analysis of the dynamics of the reaction coordinate in this simulation as well as in several other polypeptide simulations revealed that those dynamics are not diffusive, but subdiffusive. To explain these observations, we developed and tested several non-Markovian models, which include memory effects in the dynamics of the reaction coordinate in the form of time-dependent transport coefficients. We invented a novel technique, based on an overdamped generalized Langevin equation, to extract conformational memory directly from one-dimensional trajectories in single molecule experiments and simulations. Finally, we tested our theories on loop formation kinetics in intrinsically disordered proteins and found that, while mean first passage times to loop closure are well described by both one-dimensional diffusion and generalized Langevin equation, neither of them can capture long-time tails observed in distributions of transition path times in all-atom simulations.

Transition paths are fleeting events when a molecule crosses a barrier separating stable configurational basins. Recent advances in single molecule experiments, including optical tweezers-based force spectroscopy and FRET-based techniques, and data analysis methods have allowed detection of various statistical properties of transition paths. These observations have highlighted an important limitation in their current theoretical interpretation – a model of diffusive dynamics of the reaction coordinate along a one-dimensional free energy landscape is often unable to account for measurements of transition path time distributions in single molecule experiments. Here, we report similar observations in a long all-atom simulation of a small fast folding protein that exhibits multiple folding and unfolding transitions. Specifically, we discovered that the distribution of transition path times in this case is much broader than the prediction of the one-dimensional diffusion model. Moreover, direct analysis of the dynamics of the reaction coordinate in this simulation as well as in several other polypeptide simulations revealed that those dynamics are not diffusive, but subdiffusive. To explain these observations, we developed and tested several non-Markovian models, which include memory effects in the dynamics of the reaction coordinate in the form of time-dependent transport coefficients. We invented a novel technique, based on an overdamped generalized Langevin equation, to extract conformational memory directly from one-dimensional trajectories in single molecule experiments and simulations. Finally, we tested our theories on loop formation kinetics in intrinsically disordered proteins and found that, while mean first passage times to loop closure are well described by both one-dimensional diffusion and generalized Langevin equation, neither of them can capture long-time tails observed in distributions of transition path times in all-atom simulations.

Abstract: The interactions between polymers and solid-water interfaces involve various interesting physical processes, which are fundamental questions for both scientific research and a variety of practical applications, such as underwater adhesives, anti-fouling and stabilization of interested materials/particles. This study focuses on investigating the fundamental single-molecule interactions between polymers and solid surfaces in an aqueous environment using Single Molecule Force Spectroscopy (SMFS). With the help of stimuli-responsive oligo ethylene glycol copolymer, the influence of polymer hydrophobicity on single-molecule adhesion force was studied using one polymer-substrate pair. By changing the NaCl concentration, the polymer underwent a transition from a hydrophilic to a hydrophobic state due to the suppressed hydration of side chains. It was found that the single-molecule adhesion force on a MoS2 basal surface in the presence of 1 mM NaCl was around 53 pN. The force almost remained constant in low NaCl concentration up to 0.5 M, followed by a significant increase with increasing NaCl concentration before approaching a plateau at NaCl concentration as high as 1 M, which indicated that the single-molecule adhesion force was sensitive to polymer hydrophobicity. To further understand the probed dependence of single-molecule force on polymer hydrophobicity, the anisotropic property of MoS2 was utilized to separate the contributions of electric double layer interaction (EDL), van der Waals interaction (vdW) and hydrophobic attraction from total single-molecule interactions. The observed pH-independent force excludes the contribution of EDL to the total single-molecule interaction. Compared with the hydrophobic basal surface of MoS2, the single-molecule adhesion force between the hydrophilic edge surface of MoS2 and the studied copolymer was found to be smaller than the lower detection limit of SMFS (9 pN). Thus, the vdW interaction was demonstrated to play a minor role in single-molecule interaction between the studied polymer and MoS2 surface. The overall investigations ruled out the dominant role of EDL and vdW interactions on single-molecule interactions and illustrated that the hydrophobic attraction might be the main driving force of single-molecule interaction between hydrophobic polymers and surfaces. To capture the underlying physics, a simple thermodynamic model was derived in the third part of this study. Based on the law of energy conservation, the energy inputted into the system by peeling the polymer chain from a solid-water interface under external force was related to the change of entropic free energy and adhesion free energy of the polymer chain. As the driving force holding the polymer on the MoS2-water interface was demonstrated to be mainly hydrophobic attraction, in common with other investigations, the adhesion free energy was quantified using hydrophobic hydration free energy. The single-molecule forces of polymers on MoS2 and highly oriented pyrolytic graphite (HOPG) surfaces were used to test the validity of the derived model. The good agreement between the experimental data and theoretical model further confirmed the dominant role of hydrophobic attraction in single-molecular interactions between polymers and hydrophobic surfaces. In the fourth part of this thesis, the in-depth understanding of SMFS investigation between different polymers and MoS2 surfaces was used to navigate the structure design of polymers for MoS2 exfoliation. The performance of MoS2 for various applications can be significantly improved when exfoliated into a single-layer. The key for the efficient aqueous exfoliation relies on the proper selection of polymer that is able to functionalize and stabilize the exfoliated MoS2 nanosheets in water. Therefore, the understanding and quantification of polymer-solid interaction in water is highly desired for the selection of suitable organic functionalization agents. Among the polymers studied, cationic poly (vinylbenzyl trimethyl ammonium chloride) (PVBTA) was indicated to be promising for efficient exfoliation of MoS2 due to its good water compatibility and strong interaction with both the basal and edge surfaces of MoS2. Compared with previously reported values, scaled production of single-layer MoS2 nanosheet exfoliated by the selected polymer was achieved to be one magnitude higher concentration using magnitude less treatment time. These results demonstrated that the insights from SMFS investigation is highly effective to optimize the functionalization and stabilization of interested materials in practical applications. To be noted, this study provided an efficient approach to discover appropriate surface active polymer for effective functionalization of MoS2 and could be applied in a variety of practical applications demanding optimization of materials, including underwater adhesion, dispersion or functionalization.

All biological motion is ultimately driven by molecular scale interactions and forces. Only in recent years have technologies and techniques been developed capable of probing interactions at forces of nanoNewtons to picoNewtons, and at length scales of Microns to Angstroms. However, these techniques (such as Optical Traps, Magnetic Tweezers, and Atomic Force Microscopy) are limited in their potential applications by their physical size and low experimental throughput. This dissertation presents the development of a DNA Origami based nanosensor, specifically designed to perform high-throughput, single-molecule force spectroscopy measurements. DNA Origami is a nanofabrication process driven by the specificity of Watson-Crick nucleobase pairing. By engineering the sequence of a series of oligonucleotides, Megadalton scale assemblies can be assembled through a molecular self-assembly process at the nanoscale. Here, we exploit this technique to utilize the entropic elasticity of the DNA molecule, generating a force on a bimolecular interaction the nanostructure facilitates. This interaction is monitored via an optical technique, Forster Resonance Energy Transfer, enabling high throughput and multiplexed measurements of biomolecular binding kinetics. Additionally, specific control over spatial arrangement and stiffness of these DNA Origami nanostructures makes them useful as nanoscale measurement tools. We have designed a second nanostructure to explore the role of local ligand flexibility on the strength of cellular signaling. A DNA Origami nanostructure was developed – A nanoscale platform incorporating biomolecular linker DNA strands of varying length and stiffness. Antibodies specific to a transmembrane receptor expressed on B cells were affixed to these nanostructures, and the complexes used to probe the parameters governing cellular signaling. However, traditionally DNA Origami is limited by small physical production scales, high costs, and sophisticated fabrication requirements. Here, we developed a methodology to dramatically grow the scale of DNA origami production, increase efficiency of nanostructure generation, and eliminate the need for expensive and specialized equipment typically used for fabrication (PCR Machine). As an ancillary benefit, this process also enabled generation of fabrication of DNA origami nanostructures using only rudimentary equipment as might be found in a high school or undergraduate teaching laboratory. This, in conjunction with the rapid folding our research enabled, allows DNA origami learning modules to be developed and implemented in these settings.

Mechanical forces and torques play an important role in many biological processes ranging from gene transcription to protein conformational dynamics, and intracellular transportation. A range of spectroscopy techniques have been developed for direct measurements of force and torque applied to biomolecules, and fluorescence microscopy have been used extensively to study the dynamics of biomolecules and their interactions. However, to apply both force and torque simultaneously in electromagnetic tweezers, with two components decoupled and controlled independently, has not been accomplished. Also, the combination of torque spectroscopy with fluorescence microscopy has not been explored. The goal of this dissertation was to develop a novel magnetic trapping platform that is capable of simultaneously exerting and monitoring controlled forces and torques on single biomolecules via micrometer-sized magnetic particles while recording fluorescence images with high spatial and temporal resolution. Towards this goal, a system using electromagnets composed of a monopole that generates force, and a set of quadrupoles that generate torque, was integrated into an optical microscope. With this system, it is possible to apply tension in the piconewton force range to single molecules, and to independently modulate the trap torsional stiffness to twist molecules. Secondly, a novel method has been developed to directly track the angular displacement of optically anisotropic particles undergoing rotational motion using optoelectronic detectors. This method makes image acquisition and processing procedures commonly used in previous studies for detecting rotational motion unnecessary, and, when combined with the electromagnetic tweezers, can extend the bandwidth of control and detection of mechanical states of single molecules to the kilohertz regime. Finally, a dual-color, bifocal imaging system was implemented to allow detection of spectrally and spatially distinct fluorescent particles using a single camera with sub-pixel registration between two separate channels, and low mechanical drift. In conclusion, the advancement in the force/torque spectroscopy and fluorescence imaging techniques presented here holds significant potential in biological investigations that require fast control and detection of mechanical states of molecules and their dynamic properties. This system can be applied to studies of DNA-protein interactions, DNA conformation dynamics, and their dependence on the mechanical states of the DNA molecules.

Abstract: Intrinsically disordered proteins (IDPs) represent a large subpopulation of the proteome, and are characterized by high structural plasticity and a predisposition to aggregate. These aggregates can accumulate inside or outside the cell, often forming amyloid plaques that are implicated in a variety of maladies, including Alzheimer’s disease, type II diabetes and Parkinson’s disease. Despite many advances in elucidating aggregation mechanisms, much is still unknown about the early oligomeric states, which are the suspected toxic agents in disease, since their transient nature makes them hard to study with traditional experimental methods. Here we have investigated the initial stages of aggregation of one such IDP, α-synuclein, a protein that is involved in Parkinson’s disease and other related dementias. To do so, we developed assays for characterizing the transient structure, stability, and kinetics of monomers and oligomers using optical tweezers with high spatial and temporal resolution. Measurements of the molecular extension as the proteins unfolded under tension revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Moreover, our data also revealed rapid fluctuations at low force, arising from the folding of two different classes of structure that were only marginally stable. The energy landscape for these transitions was characterized via the force-dependent kinetics derived from correlation analysis of the extension trajectories. The barriers were small, only a few kBT, but the diffusion was slow, revealing a landscape that is flat but rough. This thesis presents the first measurements of α-synuclein using optical tweezers, and also provides the first experimentally reconstructed energy landscape for an IDP.

Due to their therapeutic applications such as radical scavenging, MRI contrast imaging, Photoluminescence imaging, drug delivery, etc., nanoparticles (NPs) have a significant importance in bio-nanotechnology. The reason that prevents the utilizing NPs for drug delivery in medical field is mostly due to their biocompatibility issues (incompatibility can lead to toxicity and cell death). Changes in the surface conditions of NPs often lead to NP cytotoxicity. Investigating the role of NP surface properties (surface charges and surface chemistry) on their interactions with biomolecules (Cells, protein and DNA) could enhance the current understanding of NP cytotoxicity. Hence, it is highly beneficial to the nanotechnology community to bring more attention towards the enhancement of surface properties of NPs to make them more biocompatible and less toxic to biological systems. Surface functionalization of NPs using specific ligand biomolecules have shown to enhance the protein adsorption and cellular uptake through more favorable interaction pathways. Cerium oxide NPs (CNPs also known as nanoceria) are potential antioxidants in cell culture models and understanding the nature of interaction between cerium oxide NPs and biological proteins and cells are important due to their therapeutic application (especially in site specific drug delivery systems). The surface charges and surface chemistry of CNPs play a major role in protein adsorption and cellular uptake. Hence, by tuning the surface charges and by selecting proper functional molecules on the surface, CNPs exhibiting strong adhesion to biological materials can be prepared. By probing the nanoscale interaction forces acting between CNPs and protein molecules using Atomic Force Microscopy (AFM) based force-distance (F-D) spectroscopy, the mechanism of CNP-protein adsorption and CNP cellular uptake can be understood more quantitatively. The work presented in this dissertation is based on the application of AFM in studying the interaction forces as well as the mechanical properties of nanobiomaterials. The research protocol employed in the earlier part of the dissertation is specifically aimed to understand the operation of F-D spectroscopy technique. The elastic properties of thin films of silicon dioxide NPs were investigated using F-D spectroscopy in the high force regime of few 100 nN to 1 µN. Here, sol-gel derived porous nanosilica thin films of varying surface morphology, particle size and porosity were prepared through acid and base catalyzed process. AFM nanoindentation experiments were conducted on these films using the F-D spectroscopy mode and the nanoscale elastic properties of these films were evaluated. The major contribution of this dissertation is a study exploring the interaction forces acting between CNPs and transferrin proteins in picoNewton scale regime using the force-distance spectroscopy technique. This study projects the importance of obtaining appropriate surface charges and surface chemistry so that the NP can exhibit enhanced protein adsorption and NP cellular uptake.