Studies of Protein-Protein and Protein-Water Interactions by Small Angle X-Ray Scattering, Terahertz Spectroscopy, ASMOS, And Computer Simulation

The protein folding problem has been one of the most challenging subjects in biological physics due to its complexity. Energy landscape theory based on statistical mechanics provides a thermodynamic interpretation of the protein folding process. We have been working to answer fundamental questions about protein-protein and protein-water interactions, which are very important for describing the energy landscape surface of proteins correctly. At first, we present a new method for computing protein-protein interaction potentials of solvated proteins directly from SAXS data. An ensemble of proteins was modeled by Metropolis Monte Carlo and Molecular Dynamics simulations, and the global X-ray scattering of the whole model ensemble was computed at each snapshot of the simulation. The interaction potential model was optimized and iterated by a Levenberg-Marquardt algorithm. Secondly, we report that terahertz spectroscopy directly probes hydration dynamics around proteins and determines the size of the dynamical hydration shell. We also present the sequence and pH-dependence of the hydration shell and the effect of the hydrophobicity. On the other hand, kinetic terahertz absorption (KITA) spectroscopy is introduced to study the refolding kinetics of ubiquitin and its mutants. KITA results are compared to small angle X-ray scattering, tryptophan fluorescence, and circular dichroism results. We propose that KITA monitors the rearrangement of hydrogen bonding during secondary structure formation. Finally, we present development of the automated single molecule operating system (ASMOS) for a high throughput single molecule detector, which levitates a single protein molecule in a 10 µm diameter droplet by the laser guidance. I also have performed supporting calculations and simulations with my own program codes.

Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion

This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. For example, exocytosis, fertilization of an egg by a sperm and communication between neurons are a few among many processes that rely on some form of fusion. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) play a central role in fusion processes which is also regulated by many accessory proteins, such as synaptotagmin, complexin and Munc18. By a new lipid mixing method at the single-vesicle level, we are able to accurately detect different stages of SNARE-mediated membrane fusion including docking, hemi and full fusion via FRET value of single donor/acceptor vesicle pair. Through this single-vesicle lipid mixing assay, we discovered the vesicle aggregation induced by C2AB/Ca2+, the dual function of complexin, and the fusion promotion role of Munc18/SNARE-core binding mode. While this new method provides the information regarding the extent of the ensemble lipid mixing, the fusion pore opening between two vesicular cavities and the interaction between proteins cannot be detected. In order to overcome these limitations, we then developed a single-vesicle content mixing method to reveal the key factor of pore expansion by detecting the FRET change of dual-labeled DNA probes encapsulated in vesicles. Through our single-vesicle content mixing assay, we found the fusion pore expansion role of yeast SNAREs as well as neuronal SNAREs plus synaptotagmin 1.

Nietzsche's orphans: music and the search for unity in revolutionary Russia, 1905-1921

At the dawn of the twentieth century, Imperial Russia was in the throes of immense social, political and cultural upheaval. The effects of rapid industrialization, rising capitalism and urbanization, as well as the trauma wrought by revolution and war, reverberated through all levels of society and every cultural sphere. In the aftermath of the 1905 revolution, amid a growing sense of panic over the chaos and divisions emerging in modern life, a portion of Russian educated society (obshchestvennost’) looked to the transformative and unifying power of music as a means of salvation from the personal, social and intellectual divisions of the contemporary world. Transcending professional divisions, these “orphans of Nietzsche” comprised a distinct aesthetic group within educated Russian society. While lacking a common political, religious or national outlook, these philosophers, poets, musicians and other educated members of the upper and middle strata were bound together by their shared image of music’s unifying power, itself built upon a synthesis of Russian and European ideas. They yearned for a “musical Orpheus,” a composer capable of restoring wholeness to society through his music. My dissertation is a study in what I call “musical metaphysics,” an examination of the creation, development, crisis and ultimate failure of this Orphic worldview. To begin, I examine the institutional foundations of musical life in late Imperial Russia, as well as the explosion of cultural life in the aftermath of the 1905 Revolution, a vibrant social context which nourished the formation of musical metaphysics. From here, I assess the intellectual basis upon which musical metaphysics rested: central concepts (music, life-transformation, theurgy, unity, genius, nation), as well as the philosophical heritage of Nietzsche and the Christian thinkers Vladimir Solov’ev, Aleksei Khomiakov, Ivan Kireevskii and Lev Tolstoi. Nietzsche’s orphans’ struggle to reconcile an amoral view of reality with a deeply felt sense of religious purpose gave rise to neo-Slavophile interpretations of history, in which the Russian nation (narod) was singled out as the savior of humanity from the materialism of modern life. This nationalizing tendency existed uneasily within the framework of the multi-ethnic empire. From broad social and cultural trends, I turn to detailed analysis of three of Moscow’s most admired contemporary composers, whose individual creative voices intersected with broader social concerns. The music of Aleksandr Scriabin (1871-1915) was associated with images of universal historical progress. Nikolai Medtner (1879-1951) embodied an “Imperial” worldview, in which musical style was imbued with an eternal significance which transcended the divisions of nation. The compositions of Sergei Rachmaninoff (1873-1943) were seen as the expression of a Russian “national” voice. Heightened nationalist sentiment and the impact of the Great War spelled the doom of this musical worldview. Music became an increasingly nationalized sphere within which earlier, Imperial definitions of belonging grew ever more problematic. As the Germanic heritage upon which their vision was partially based came under attack, Nietzsche’s orphans found themselves ever more divided and alienated from society as a whole. Music’s inability to physically transform the world ultimately came to symbolize the failure of Russia’s educated strata to effectively deal with the pressures of a modernizing society. In the aftermath of the 1917 revolutions, music was transformed from a symbol of active, unifying power into a space of memory, a means of commemorating, reinterpreting, and idealizing the lost world of Imperial Russia itself.

Heat dissipation from carbon nano-electronics

The incorporation of graphitic compounds such as carbon nanotubes (CNTs) and graphene into nano-electronic device packaging holds much promise for waste heat management given their high thermal conductivities. However, as these graphitic materials must be used in together with other semiconductor/insulator materials, it is not known how thermal transport is affected by the interaction. Using different simulation techniques, in this thesis, we evaluate the thermal transport properties - thermal boundary conductance (TBC) and thermal conductivity - of CNTs and single-layer graphene in contact with an amorphous SiO2 (a-SiO2) substrate. First, the theoretical methodologies and concepts used in our simulations are presented. In particular, two concepts are described in detail as they are necessary for the understanding of the subsequent chapters. The first is the linear response Green-Kubo (GK) theory of thermal boundary conductance (TBC), which we develop in this thesis, and the second is the spectral energy density method, which we use to directly compute the phonon lifetimes and thermal transport coefficients. After we set the conceptual foundations, the TBC of the CNT-SiO2 interface is computed using non- equilibrium molecular dynamics (MD) simulations and the new Green-Kubo method that we have developed. Its dependence on temperature, the strength of the interaction with the substrate, and tube diameter are evaluated. To gain further insight into the phonon dynamics in supported CNTs, the scattering rates are computed using the spectral energy density (SED) method. With this method, we are able to distinguish the different scattering mechanisms (boundary and CNT-substrate phonon-phonon) and rates. The phonon lifetimes in supported CNTs are found to be reduced by contact with the substrate and we use that lifetime reduction to determine the change in CNT thermal conductivity. Next, we examine thermal transport in graphene supported on SiO2. The phonon contribution to the TBC of the graphene-SiO2 interface is computed from MD simulations and found to agree well with experimentally measured values. We derive the theory of remote phonon scattering of graphene electrons and compute the heat transfer coefficient dependence on doping level and temperature. The thermal boundary conductance from remote phonon scattering is found to be an order of magnitude smaller than that of the phonon contribution. The in-plane thermal conductivity of supported graphene is calculated from MD simulations. The experimentally measured order of magnitude reduction in thermal conductivity is reproduced in our simulations. We show that this reduction is due to the damping of the flexural (ZA) modes. By varying the interaction between graphene and the substrate, the ZA modes hybridize with the substrate Rayleigh modes and the dispersion of the hybridized modes is found to linearize in the strong coupling limit, leading to an increased thermal conductance in the composite structure.

Bidirectional cargo transport by microtubule-based molecular motors

We have reconstituted a simple in vitro system using only mammalian dynein and mammalian kinesin attached to a single cargo. These cargoes undergo saltatory motion typically seen in vivo, indicating that the motors engage in a tug-of-war. When the complex hits a barrier, the cargo often reverses direction. In some cases, it tries several up-and-back motions, during which time the dynein likely pulls the cargo onto a different protofilament, and is sometimes able to bypass the blockage. This explains why eliminating kinesin or dynein stops motion in both directions in vivo. We also find that mammalian dynein, but not kinesin, often takes backwards steps when under backward force. However, yeast dynein coupled with mammalian kinesin does not display these attributes, as expected.