The stability of myosin subfragment 2 was analyzed using gravitational force spectroscopy. The region was found to destabilize under physiological force loads, indicating the possibility that subfragment 2 may uncoil to facilitate actin binding during muscle contraction. As a control, synthetic cofilaments were produced to discover if the observations in the single molecule assay were due to the lack of the stability provided by the thick filament. Statistically, there was no difference between the single molecule assay data and the synthetic cofilament assay data. Thus, the instability of the region is due to intrinsic properties within subfragment 2.

<p>This research describes the development of a state-of-the-art atomic force microscope (AFM) for improved force spectroscopy. Although the AFM has been used extensively in this field of research, the performance of the instrument has been limited by inefficient operation techniques, incorrect experimental assumptions, and inadequate controller design. This research focuses on overcoming these deficiencies by providing precise control over the instrument for specialized research in a manner that is conducive to the natural science researcher.</p><p>To facilitate this research, a custom, multi-axis AFM system was constructed. The instrument was designed primarily for AFM-based force spectroscopy and as a result a substantial amount of research focused on the development of a wide variety of approach/retraction methods for the instrument. Defining research in this area included the development of methods to minimize potentially damaging compressive forces, form polymer bridges at different tip-sample gap widths, produce clean, deconvoluted force-extension curves, and limit single molecule force spectroscopy pulling geometry errors. In an effort to increase the efficiency of the instrument, the programs developed during this research were fully automated, allowing autonomous operation of the instrument for long periods of time. To compliment the data collection programs, both manual and automated analysis programs with force-volume imaging capabilities were also developed.</p><p>By studying the AFM from a dynamic systems, measurements, and controls approach, the resulting controllers were tailored to meet the process requirements of the intended applications. In doing so, the sensitivity of the instrument was improved for applications of interest. By incorporating control over the environment, contact force, loading rate, and pulling angle, the research has increased the accuracy of the AFM such that molecules and receptor-ligand binding events can be investigated with greater detail. Furthermore, the incorporation of a graphical user interface and automated data collection and analysis tools has made the AFM a more user-friendly, efficient instrument for the natural science researcher.</p> / Dissertation

No description available.

Yes / We combine single molecule force spectroscopy measurements with all-atom metadynamics simulations to investigate the cross-materials binding strength trends of DNA fragments adsorbed at the aqueous graphite C(0001) and Au(111) interfaces. Our simulations predict this adsorption at the level of the nucleobase, nucleoside, and nucleotide. We find that despite challenges in making clear, careful connections between the experimental and simulation data, reasonable consistency between the binding trends between the two approaches and two substrates was evident. On C(0001), our simulations predict a binding trend of dG > dA ≈ dT > dC, which broadly aligns with the experimental trend. On Au(111), the simulation-based binding strength trends reveal stronger adsorption for the purines relative to the pyrimadines, with dG ≈ dA > dT ≈ dC. Moreover, our simulations provide structural insights into the origins of the similarities and differences in adsorption of the nucleic acid fragments at the two interfaces. In particular, our simulation data offer an explanation for the differences observed in the relative binding trend between adenosine and guanine on the two substrates.

Master of Science / Department of Physics / Robert Szoszkiewicz / For proteins in living cells, forces are present at all levels. These range from macroscopic to single molecule levels. Single molecule atomic force microscopy (AFM) in force extension (FX) and force clamp (FC) modes can investigate the mechanical properties of proteins, for example, forces at which proteins unfold, or the kinetics of these processes. In the FX-AFM experiments, proteins are pulled at constant velocity, while in FC-AFM experiments, proteins are pulled at constant force. This thesis describes i) how a single molecule FX/FC-AFM was constructed using various components, ii) how it was calibrated and tested using (I27)4 polyprotein, and iii) how it was applied to the studies of a Notch construct. Building up the single molecule FX/FC-AFM system opened a path to investigate the mechanical properties of proteins. Such a system was tested on a known protein construct, hence the usage of the (I27)4 polyprotein. The Notch protein is a signaling protein that plays a role in triggering breast cancer. It is believed that understanding the mechanical properties of Notch can help to understand its oncogenic functions. We have successfully constructed and calibrated the FX/FC-AFM setup. It was found that the AFM worked for the standard calibration protein of (I27)4. The results on a Notch construct revealed our ability to see some conformational transition state in this molecule under force. These results opened a path for further investigations of a Notch construct at various physiologically relevant conditions.

The focus of this dissertation is on refining atomic force micrscopy (AFM) methods and data analysis routines to measure the viscoelastic mechanical properties of soft polymer and biological materials in relevant fluid environments and in vivo using a range of relevant temperatures, applied forces, and loading rates. These methods are directly applied here to a several interesting synthetic and biological materials. First, we probe poly(n-butyl methacrylate) (PnBMA), above, at and below its glass transition temperature in order to verify our experimental procedure. Next, we use AFM to study the viscoelastic properties of coating materials and additives of silicone-based soft contact lenses in a tear-like saline solution. Finally, a major focus in this dissertation is determining the fundamental mechanical properties that contribute to the excellent sensitivity of the strain sensing organs in a wandering spider (Cupiennius salei) by probing under in vivo conditions. These strain-sensing organs are known to have a significant viscoelastic component. Thus, the cuticle of living spiders is directly investigated in near-natural environments (high humidity, temperatures from 15-40 °C). The main achievements of these studies can be summarized through the following findings: We suggest that full time-temperature-modulus relationships are necessary for the understanding of soft materials systems, and present a practical method for obtaining such relationships. These studies will have a direct impact on both scientists in the metrology field by developing practical experimental procedures and data analysis routines to investigate viscoelastic mechanical properties at the nanoscale, and future materials scientists and engineers by showing via spider mechanosensory systems how viscoelasticity can be applied for functional use in sensing technology.

Understanding the intermolecular forces and dynamics of insulin self-assembly is crucial for devising formulations for the treatment of insulin-dependent diabetes. Insulin must dissociate from its hexameric storage form, through an intermediate dimer form, to the bioactive monomer before receptor binding. Specifically, the dimer dissociation is a pivotal step to control insulin dynamics and self-assembly. Steered molecular dynamics simulations were performed on native insulin to provide molecular insight into the insulin dissociation force spectroscopy experiment. Our simulation results of force-induced dimer dissociation revealed that the dimer dissociation occurs near the limit of extensibility of the B-chain with significant conformational changes to the monomer(s). These long-range interactions, consistent with our experiments, are due to stronger inter-monomer interactions across the anti-parallel β-sheet interface than any other intra-monomer interaction. Novel atomistic data played an important role in detailed structural characterization of multiple unfolding and dissociation pathways that depend on the relative strength of the inter-monomer interactions and the intra-monomer interactions. Comparative simulations of two rapid-acting insulin analogues (LysB28ProB29, AspB28) to native insulin were performed to investigate the effect of sequence on the dimer dissociation. The hypothesis is that site-specific alterations to the dimer-forming surface of two rapid-acting analogues will result in a weakening of the inter-monomer interactions, which would be reflected during force-induced dimer dissociation. The results revealed that these analogues dissociates with lower probability of long-range interactions and a corresponding reduction in B-chain extension. B-chain extensibility is thus a characteristic marker of inter-monomer interactions and multiple unfolding pathways. These data agree with the design strategies of sequence modifications to the weakened inter-monomer interface applied to the synthesis of rapid-acting insulin analogues. In contrast, the ligand-induced alteration to the strengthened inter-monomer interactions through a specific GluB13s-zinc bridge contributed to the unique unfolding force curves, so it can be applicable as design strategy to the development of a novel long-acting analogue. Overall, our force spectroscopy studies on insulin native and analogues have successfully provided atomistic insights into the dimer dissociation characteristics and control strategies of self-assembly. In addition, this study would provide a framework for the structure-dynamics-function relationships of insulin-insulin receptor binding.

Understanding the intermolecular forces and dynamics of insulin self-assembly is crucial for devising formulations for the treatment of insulin-dependent diabetes. Insulin must dissociate from its hexameric storage form, through an intermediate dimer form, to the bioactive monomer before receptor binding. Specifically, the dimer dissociation is a pivotal step to control insulin dynamics and self-assembly. Steered molecular dynamics simulations were performed on native insulin to provide molecular insight into the insulin dissociation force spectroscopy experiment. Our simulation results of force-induced dimer dissociation revealed that the dimer dissociation occurs near the limit of extensibility of the B-chain with significant conformational changes to the monomer(s). These long-range interactions, consistent with our experiments, are due to stronger inter-monomer interactions across the anti-parallel β-sheet interface than any other intra-monomer interaction. Novel atomistic data played an important role in detailed structural characterization of multiple unfolding and dissociation pathways that depend on the relative strength of the inter-monomer interactions and the intra-monomer interactions. Comparative simulations of two rapid-acting insulin analogues (LysB28ProB29, AspB28) to native insulin were performed to investigate the effect of sequence on the dimer dissociation. The hypothesis is that site-specific alterations to the dimer-forming surface of two rapid-acting analogues will result in a weakening of the inter-monomer interactions, which would be reflected during force-induced dimer dissociation. The results revealed that these analogues dissociates with lower probability of long-range interactions and a corresponding reduction in B-chain extension. B-chain extensibility is thus a characteristic marker of inter-monomer interactions and multiple unfolding pathways. These data agree with the design strategies of sequence modifications to the weakened inter-monomer interface applied to the synthesis of rapid-acting insulin analogues. In contrast, the ligand-induced alteration to the strengthened inter-monomer interactions through a specific GluB13s-zinc bridge contributed to the unique unfolding force curves, so it can be applicable as design strategy to the development of a novel long-acting analogue. Overall, our force spectroscopy studies on insulin native and analogues have successfully provided atomistic insights into the dimer dissociation characteristics and control strategies of self-assembly. In addition, this study would provide a framework for the structure-dynamics-function relationships of insulin-insulin receptor binding.

This thesis addresses development of a new force spectroscopy tool, correlation force spectroscopy (CFS), for the measurement of the mechanical properties of very small volumes of material (molecular to µm³) at kHz-MHz time-scales. CFS is based on atomic force microscopy (AFM) and the principles of CFS resemble those of dual-trap optical tweezers. CFS consists of two closely-spaced micro-cantilevers that undergo thermal fluctuations. Measurement of the correlation in thermal fluctuations of the two cantilevers can be used to determine the mechanical properties of the soft matter, e.g. a polymeric molecule, that connects the gap between the two cantilevers. Modeling of the correlations yields the effective stiffness and damping of the molecule. The resolution in stiffness is limited by the stiffness of the cantilever and the frequency by the natural frequency of the cantilevers, but, importantly, the damping resolution is not limited by the damping of the cantilever, which has enabled high-resolution measurements of the internal friction of a polymer. The concept of CFS was originally presented by Roukes' group in Caltech [Arlett et al., Lecture Notes in Physics, 2007]; I developed the first practical versions of CFS for experimentation, and have used it in two applications (1) microrheology of Newtonian fluids and (2) single molecule force spectroscopy. To understand the correlation in thermal fluctuations of two cantilevers I initially validated the theoretical approach for analyzing correlation in terms of deterministic model using the fluctuation-dissipation theorem [Paul and Cross, PRL, 2004]. I have shown that the main advantages of such correlation measurements are a large improvement in the ability to resolve stiffness and damping. Use of CFS as a rheometer was validated by comparison between experimental data and finite element modeling of the deterministic vibrations of the cantilevers using the known viscosity and density of fluids. Work in this thesis shows that the data can also be accurately fitted using a simple harmonic oscillator model, which can be used for rapid rheometric measurements, after calibration. The mechanical properties of biomolecules such as dextran and single stranded DNA (ssDNA) are also described. CFS measurements of single molecule properties of ssDNA reveal the internal friction of the molecule in solution. / Ph. D.

Myosin subfragment-2 (S2) is an intrinsically unstable coiled coil. This dissertation tests if the mechanical stability of myosin S2 would influence the availability of myosin S1 heads to actin thin filaments. The elevated instability in myosin S2 coiled coil could be one of the causes for hypercontractility in Familial Hypertrophic Cardiomyopathy (FHC). As hypothesized FHC mutations, namely E924K and E930del, in myosin S2 displayed an unstable myosin S2 coiled coil compared to wild type as measured by Fluorescence Resonant Energy Transfer (FRET) and gravitational force spectroscopy (GFS). To remedy this, anti-S2 peptides; the stabilizer and the destabilizer peptides by namesake were designed in our lab to increase and decrease the stability of myosin S2 coiled coil to influence the actomyosin interaction. Firstly, the effectiveness of anti-S2 peptides were tested on muscle myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by the stabilizer and decreased by the destabilizer across the cardiac and skeletal myosin S2 isoform but not for the smooth muscle isoform. The destabilizer peptide had dissociation binding constants of 9.97 × 10-1 μM to MYH7 isoform, 1.00 μM to MYH2 isoform, and no impact on MYH11, and the stabilizer peptide had dissociation binding constants of 2.12 × 10-2 μM to MYH7 isoform, 3.41 × 10-1 μM to MYH2 isoform, and no impact on MYH11 revealed by FRET. In presence of the stabilizer, FRET assay, affinity of the E930del and E924K increased by 10.23 and 0.60 fold respectively. The force required to uncoil muscle myosin S2 peptides in the presence of the stabilizer peptide was more than in its absence in muscle myosin S2 isoforms of MYH7 (1.80 fold higher), MYH2 (1.40 fold higher), and E930del (2.60 fold higher) and no change for MYH11 compared to control. The force required to uncoil muscle myosin S2 in presence of the destabilizer was less than in its absence in both MYH7 (2.00 fold lower) and MYH2 (2.5 fold lower) but the same for MYH11 compared to their controls. Both FRET and GFS assays demonstrated that both anti-S2 peptides do not have any impact on smooth muscle myosin S2 isoform. In FRET assay, there was no significant difference in the lifetime value in the presence or absence of anti-S2 peptides in smooth muscle myosin S2. In GFS assay, there was no significant difference in the force required to uncoil the dimer in presence or absence of the anti-S2 peptides smooth muscle myosin S2. Effectively, the stabilizer peptide improved the stability of FHC mutant (E924K and E930del) myosin S2 peptide. FHC mutations showed high lifetime value in FRET assay and low force to uncoil coiled coil myosin S2 in GFS assay. In the presence of the stabilizer, lifetime value decreased in FRET assay and more force was required to uncoil myosin S2 coiled coil in GFS assay. This study demonstrated that structure of muscle myosin S2 can be altered by small peptides. The stabilizer peptide enhanced dimer formation in wild type and mutant cardiac, and skeletal myosin S2 peptides, and destabilizer increased flexibility of cardiac and skeletal myosin S2 wild type peptide. Neither anti-S2 peptides had impacts on smooth muscle myosin S2 isoform. The study thus effectively demonstrates the mechanical stability of myosin S2 coiled coil in striated muscle system could be modified using the specific anti-S2 peptides. Stabilizer of the anti-S2 peptide was effective to remedy the dampened stability of FHC myosin S2 coiled coil thus providing a new dimension of treating cardiovascular and skeletal muscle disorders by targeting the structural property of muscle proteins.