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Sec-facilitated Protein Translocation and Membrane Integration
Get PDFThe Sec translocon is a central component of the cellular machinery for targeting and delivering nascent proteins. Ubiquitous across all kingdoms of life, it is a protein-conducting channel that facilitates recognition of integral membrane protein domains and the establishment of integral membrane protein topology. Structural, biochemical, and biophysical studies have illuminated the role of the Sec translocon in both cotranslational and posttranslational protein targeting. In particular, quantitative assays have established the dependence of transmembrane domain (TM) stop-transfer efficiency and integral membrane protein topogenesis on the physicochemical properties of the translocon and protein nascent chain. These studies provide a valuable starting point for understanding the molecular regulation of the translocon and its sensitivity to mutations in protein sequence and external driving forces; however, complexities associated with the Sec machinery, including the role of collaborating molecular motors, the importance of large-scale conformational changes in the translocon, and the crowded molecular environment of the channel interior, obscure the mechanistic basis for many experimentally observed trends. My PhD research has focused on the development of a unified, mechanistic understanding of Sec-facilitated protein targeting.
Using both atomistic and coarse-grained molecular simulations, we have investigated the conformational landscape for the Sec translocon. We found that inclusion of a hydrophobic peptide substrate in the translocon stabilizes an open conformation of the lateral gate (LG) that is necessary for membrane integration, whereas inclusion of a hydrophilic peptide substrate favors only the closed LG conformation. We demonstrated that the translocon plug moiety adopts markedly different conformations in the channel, depending on whether the substrate peptide is hydrophobic or hydrophilic in character. Finally, we showed that the energetics of the translocon LG opening in the presence of the substrate peptides can be modeled in terms of the energetics of the peptide interface with the membrane. The manuscript associated with this study is published in PNAS, 107, 5399 (2010).
We further developed a novel computational protocol that combines nonequilibrium growth of the nascent protein with microsecond-timescale molecular dynamics trajectories. Analysis of multiple, long-timescale simulations elucidated molecular features of protein insertion into the translocon, including signal-peptide docking at the translocon LG, large-lengthscale conformational rearrangement of the translocon LG helices, and partial membrane integration of hydrophobic nascent-protein sequences. Furthermore, the simulations demonstrated the role of specific molecular interactions in the regulation of protein secretion, membrane integration, and integral membrane protein topology. Salt-bridge contacts between the nascent-protein N-terminus, cytosolic translocon residues, and phospholipid head groups were shown to favor conformations of the nascent protein upon early-stage insertion that are consistent with the Type II (Ncyt/Cexo) integral membrane protein topology; and extended hydrophobic contacts between the nascent protein and the membrane lipid bilayer were shown to stabilize configurations that are consistent with the Type III (Nexo/Ccyt) topology. These results provide a detailed, mechanistic basis for understanding experimentally observed correlations between integral membrane protein topology, translocon mutagenesis, and nascent-protein sequence. The manuscript associated with this study is published in J. Am. Chem. Soc., 134, 13700 (2012).
Finally, we introduced a coarse-grained modeling approach that spans the nanosecond to minute-timescale dynamics of cotranslational protein translocation. The method enabled direct simulation of both integral membrane protein topogenesis and TM stop-transfer efficiency. Simulations revealed multiple kinetic pathways for protein integration, including a mechanism in which the nascent protein undergoes slow-timescale reorientation, or flipping, in the confined environment of the translocon channel. Competition among these pathways gives rise to the experimentally observed dependence of protein topology on ribosomal translation rate and protein length. We further demonstrated that sigmoidal dependence of stop-transfer efficiency on TM hydrophobicity arises from local equilibration of the TM across the translocon LG, and it was predicted that slowing ribosomal translation yields decreased stop-transfer efficiency in long proteins. This work reveals the balance between equilibrium and nonequilibrium processes in protein targeting, and it provides new insight into the molecular regulation of the Sec translocon. The manuscript associated with this study is published in Cell Reports, in press.
This research has significantly enriched the mechanistic understanding of Sec-facilitated protein translocation and membrane integration with ample molecular details. The unifying picture that we propose establishes fundamental connections between previously disparate experimental studies, and it lays down the foundation for future verification and refinement.</p
Light to Electrons to Bonds: Imaging Water Splitting and Collecting Photoexcited Electrons
Get PDFPhotoelectrochemical devices can store solar energy as chemical bonds in fuels, but more control over the materials involved is needed for economic feasibility. Both efficient capture of photon energy into electron energy and subsequent electron transfer and bond formation are necessary, and this thesis explores various steps of the process. To look at the electrochemical fuel formation step, the spatially-resolved reaction rate on a water-splitting electrode was imaged during operation at a few-micron scale using optical microscopy. One method involved localized excitation of a semiconductor photoanode and recording the growth rate of bubbles to determine the local reaction rate. A second method imaged the reactant profile with a pH-sensitive fluorophore in the electrolyte to determine the local three-dimensional pH profile at patterned electrocatalysts in a confocal microscope. These methods provide insight on surface features optimal for efficient electron transfer into fuel products.
A second set of studies examined the initial process of photoexcited electron transport and collection. An independent method to measure the minority carrier diffusion length in semiconductor photoelectrodes was developed, in which a wedge geometry is back illuminated with a small scanned spot. The diffusion length can be determined from the exponential decrease of photocurrent with thickness, and the method was demonstrated on solid-state silicon wedge diodes, as well as tungsten oxide thin-film wedge photoanodes. Finally, the possibility of absorbing and collecting sub-bandgap illumination via plasmon-enhanced hot carrier internal photoemission was modeled to predict the energy conversion efficiency. The effect of photon polarization on emission yield was experimentally tested using gold nanoantennas buried in silicon, and the correlation was found to be small.</p
Current Transport and Onset-Related Phenomena in an MPD Thruster Modified by Applied Magnetic Fields
Get PDFThis work investigated the effects of tailored, externally-applied magnetic fields on current transport and near-anode processes in the plasma discharge of a magnetoplasmadynamic thruster (MPDT). Electrical and plasma diagnostics were used to determine whether applied magnetic fields could mitigate the effects of the "onset" phenomena, including large-amplitude terminal voltage fluctuations and high anode fall voltages associated with unstable operation and anode erosion. A new MPDT was developed and operated with quasi-steady 1 ms pulses from 36 kW to 3.3 MW with argon propellant. Three magnetic configurations studied included self-field operation (without external electromagnets) and two applied poloidal magnetic fields. One configuration used magnetic field lines tangential to the anode lip (and intersecting the anode further upstream) and the other created a magnetic cusp intersecting the anode downstream.
The influence of the applied fields on the discharge current streamlines, current densities, and key plasma properties (electron temperature, number density, and plasma potential) was studied. Key findings included that the current pattern and current densities redistributed to follow the applied magnetic field lines. Also, the anode fall voltage was substantially reduced with both applied fields over a large range of currents (and eliminated at 8 kA). These results occurred because applied magnetic field lines intersecting the anode provided a high conductivity path and reduced the local electric field required to sustain the radial current densities. The applied fields reduced the amplitude and frequency of the terminal voltage fluctuations (up to 49%) over a broad range of currents and also decreased transients in the ion saturation current, which suggest reduction of current filamentation and surface-eroding anode spots. Additionally, the cusp field reduced mean terminal voltages over the entire range of discharge currents (up to 31%), and the tangential field lowered terminal voltages below 10.7 kA. These significant reductions in onset-related behaviors should lead to improved thruster lifetime and increased efficiency. These results suggest a distinctive and more effective approach to influencing the near-anode phenomena and mitigating the effects of onset with appropriately designed applied magnetic fields that differ from those used in the vast majority of conventional, so-called "applied-field MPD thrusters."</p
Searches for New Symmetries in pp Collisions with the Razor Kinematic Variables at √s = 7 TeV
Get PDFThe construction and LHC phenomenology of the razor variables MR, an event-by-event indicator of the heavy particle mass scale, and R, a dimensionless variable related to the transverse momentum imbalance of events and missing transverse energy, are presented. The variables are used in the analysis of the first proton-proton collisions dataset at CMS (35 pb-1) in a search for superpartners of the quarks and gluons, targeting indirect hints of dark matter candidates in the context of supersymmetric theoretical frameworks. The analysis produced the highest sensitivity results for SUSY to date and extended the LHC reach far beyond the previous Tevatron results. A generalized inclusive search is subsequently presented for new heavy particle pairs produced in √s = 7 TeV proton-proton collisions at the LHC using 4.7±0.1 fb-1 of integrated luminosity from the second LHC run of 2011. The selected events are analyzed in the 2D razor-space of MR and R and the analysis is performed in 12 tiers of all-hadronic, single and double leptons final states in the presence and absence of b-quarks, probing the third generation sector using the event heavy-flavor content. The search is sensitive to generic supersymmetry models with minimal assumptions about the superpartner decay chains. No excess is observed in the number or shape of event yields relative to Standard Model predictions. Exclusion limits are derived in the CMSSM framework with gluino masses up to 800 GeV and squark masses up to 1.35 TeV excluded at 95% confidence level, depending on the model parameters. The results are also interpreted for a collection of simplified models, in which gluinos are excluded with masses as large as 1.1 TeV, for small neutralino masses, and the first-two generation squarks, stops and sbottoms are excluded for masses up to about 800, 425 and 400 GeV, respectively.
With the discovery of a new boson by the CMS and ATLAS experiments in the γ-γ and 4 lepton final states, the identity of the putative Higgs candidate must be established through the measurements of its properties. The spin and quantum numbers are of particular importance, and we describe a method for measuring the JPC of this particle using the observed signal events in the H to ZZ* to 4 lepton channel developed before the discovery. Adaptations of the razor kinematic variables are introduced for the H to WW* to 2 lepton/2 neutrino channel, improving the resonance mass resolution and increasing the discovery significance. The prospects for incorporating this channel in an examination of the new boson JPC is discussed, with indications that this it could provide complementary information to the H to ZZ* to 4 lepton final state, particularly for measuring CP-violation in these decays.</p
Essays in Mechanism Design
Get PDFThis dissertation contains three essays on mechanism design. The common goal of these essays is to assist in the solution of different resource allocation problems where asymmetric information creates obstacles to the efficient allocation of resources. In each essay, we present a mechanism that satisfactorily solves the resource allocation problem and study some of its properties. In our first essay, ”Combinatorial Assignment under Dichotomous Preferences”, we present a class of problems akin to time scheduling without a pre-existing time grid, and propose a mechanism that is efficient, strategy-proof and envy-free. Our second essay, ”Monitoring Costs and the Management of Common-Pool Resources”, studies what can happen to an existing mechanism — the individual tradable quotas (ITQ) mechanism, also known as the cap-and-trade mechanism — when quota enforcement is imperfect and costly. Our third essay, ”Vessel Buyback”, coauthored with John O. Ledyard, presents an auction design that can be used to buy back excess capital in overcapitalized industries
Learning and Using Taxonomies for Visual and Olfactory Classification
Get PDFHumans are able of distinguishing more than 5000 visual categories even in complex environments using a variety of different visual systems all working in tandem. We seem to be capable of distinguishing thousands of different odors as well. In the machine learning community, many commonly used multi-class classifiers do not scale well to such large numbers of categories. This thesis demonstrates a method of automatically creating application-specific taxonomies to aid in scaling classification algorithms to more than 100 cate- gories using both visual and olfactory data. The visual data consists of images collected online and pollen slides scanned under a microscope. The olfactory data was acquired by constructing a small portable sniffing apparatus which draws air over 10 carbon black polymer composite sensors. We investigate performance when classifying 256 visual categories, 8 or more species of pollen and 130 olfactory categories sampled from common household items and a standardized scratch-and-sniff test. Taxonomies are employed in a divide-and-conquer classification framework which improves classification time while allowing the end user to trade performance for specificity as needed. Before classification can even take place, the pollen counter and electronic nose must filter out a high volume of background “clutter” to detect the categories of interest. In the case of pollen this is done with an efficient cascade of classifiers that rule out most non-pollen before invoking slower multi-class classifiers. In the case of the electronic nose, much of the extraneous noise encountered in outdoor environments can be filtered using a sniffing strategy which preferentially samples the visensor response at frequencies that are relatively immune to background contributions from ambient water vapor. This combination of efficient background rejection with scalable classification algorithms is tested in detail for three separate projects: 1) the Caltech-256 Image Dataset, 2) the Caltech Automated Pollen Identification and Counting System (CAPICS) and 3) a portable electronic nose specially constructed for outdoor use
Lattice Quantum Codes and Exotic Topological Phases of Matter
Get PDFThis thesis addresses whether it is possible to build a robust memory device for quantum information. Many schemes for fault-tolerant quantum information processing have been developed so far, one of which, called topological quantum computation, makes use of degrees of freedom that are inherently insensitive to local errors. However, this scheme is not so reliable against thermal errors. Other fault-tolerant schemes achieve better reliability through active error correction, but incur a substantial overhead cost. Thus, it is of practical importance and theoretical interest to design and assess fault-tolerant schemes that work well at finite temperature without active error correction.
In this thesis, a three-dimensional gapped lattice spin model is found which demonstrates for the first time that a reliable quantum memory at finite temperature is possible, at least to some extent. When quantum information is encoded into a highly entangled ground state of this model and subjected to thermal errors, the errors remain easily correctable for a long time without any active intervention, because a macroscopic energy barrier keeps the errors well localized. As a result, stored quantum information can be retrieved faithfully for a memory time which grows exponentially with the square of the inverse temperature. In contrast, for previously known types of topological quantum storage in three or fewer spatial dimensions the memory time scales exponentially with the inverse temperature, rather than its square.
This spin model exhibits a previously unexpected topological quantum order, in which ground states are locally indistinguishable, pointlike excitations are immobile, and the immobility is not affected by small perturbations of the Hamiltonian. The degeneracy of the ground state, though also insensitive to perturbations, is a complicated number-theoretic function of the system size, and the system bifurcates into multiple noninteracting copies of itself under real-space renormalization group transformations. The degeneracy, the excitations, and the renormalization group flow can be analyzed using a framework that exploits the spin model's symmetry and some associated free resolutions of modules over polynomial algebras.</p
Beyond Watson and Crick: Programming the Self-Assembly and Reconfiguration of DNA Nanostructures Based on Stacking Interactions
Get PDFLife is the result of the execution of molecular programs: like how an embryo is fated to become a human or a whale, or how a person’s appearance is inherited from their parents, many biological phenomena are governed by genetic programs written in DNA molecules. At the core of such programs is the highly reliable base pairing interaction between nucleic acids. DNA nanotechnology exploits the programming power of DNA to build artificial nanostructures, molecular computers, and nanomachines. In particular, DNA origami—which is a simple yet versatile technique that allows one to create various nanoscale shapes and patterns—is at the heart of the technology. In this thesis, I describe the development of programmable self-assembly and reconfiguration of DNA origami nanostructures based on a unique strategy: rather than relying on Watson-Crick base pairing, we developed programmable bonds via the geometric arrangement of stacking interactions, which we termed stacking bonds. We further demonstrated that such bonds can be dynamically reconfigurable.
The first part of this thesis describes the design and implementation of stacking bonds. Our work addresses the fundamental question of whether one can create diverse bond types out of a single kind of attractive interaction—a question first posed implicitly by Francis Crick while seeking a deeper understanding of the origin of life and primitive genetic code. For the creation of multiple specific bonds, we used two different approaches: binary coding and shape coding of geometric arrangement of stacking interaction units, which are called blunt ends. To construct a bond space for each approach, we performed a systematic search using a computer algorithm. We used orthogonal bonds to experimentally implement the connection of five distinct DNA origami nanostructures. We also programmed the bonds to control cis/trans configuration between asymmetric nanostructures.
The second part of this thesis describes the large-scale self-assembly of DNA origami into two-dimensional checkerboard-pattern crystals via surface diffusion. We developed a protocol where the diffusion of DNA origami occurs on a substrate and is dynamically controlled by changing the cationic condition of the system. We used stacking interactions to mediate connections between the origami, because of their potential for reconfiguring during the assembly process. Assembling DNA nanostructures directly on substrate surfaces can benefit nano/microfabrication processes by eliminating a pattern transfer step. At the same time, the use of DNA origami allows high complexity and unique addressability with six-nanometer resolution within each structural unit.
The third part of this thesis describes the use of stacking bonds as dynamically breakable bonds. To break the bonds, we used biological machinery called the ParMRC system extracted from bacteria. The system ensures that, when a cell divides, each daughter cell gets one copy of the cell’s DNA by actively pushing each copy to the opposite poles of the cell. We demonstrate dynamically expandable nanostructures, which makes stacking bonds a promising candidate for reconfigurable connectors for nanoscale machine parts. </p
Control of Wettability of Carbon Nanotube Array by Reversible Dry Oxidation for Superhydrophobic Coating and Supercapacitor Applications
Get PDFIn this thesis, dry chemical modification methods involving UV/ozone, oxygen plasma, and vacuum annealing treatments are explored to precisely control the wettability of CNT arrays. By varying the exposure time of these treatments the surface concentration of oxygenated groups adsorbed on the CNT arrays can be controlled. CNT arrays with very low amount of oxygenated groups exhibit a superhydrophobic behavior. In addition to their extremely high static contact angle, they cannot be dispersed in DI water and their impedance in aqueous electrolytes is extremely high. These arrays have an extreme water repellency capability such that a water droplet will bounce off of their surface upon impact and a thin film of air is formed on their surface as they are immersed in a deep pool of water. In contrast, CNT arrays with very high surface concentration of oxygenated functional groups exhibit an extreme hydrophilic behavior. In addition to their extremely low static contact angle, they can be dispersed easily in DI water and their impedance in aqueous electrolytes is tremendously low. Since the bulk structure of the CNT arrays are preserved during the UV/ozone, oxygen plasma, and vacuum annealing treatments, all CNT arrays can be repeatedly switched between superhydrophilic and superhydrophobic, as long as their O/C ratio is kept below 18%.
The effect of oxidation using UV/ozone and oxygen plasma treatments is highly reversible as long as the O/C ratio of the CNT arrays is kept below 18%. At O/C ratios higher than 18%, the effect of oxidation is no longer reversible. This irreversible oxidation is caused by irreversible changes to the CNT atomic structure during the oxidation process. During the oxidation process, CNT arrays undergo three different processes. For CNT arrays with O/C ratios lower than 40%, the oxidation process results in the functionalization of CNT outer walls by oxygenated groups. Although this functionalization process introduces defects, vacancies and micropores opening, the graphitic structure of the CNT is still largely intact. For CNT arrays with O/C ratios between 40% and 45%, the oxidation process results in the etching of CNT outer walls. This etching process introduces large scale defects and holes that can be obviously seen under TEM at high magnification. Most of these holes are found to be several layers deep and, in some cases, a large portion of the CNT side walls are cut open. For CNT arrays with O/C ratios higher than 45%, the oxidation process results in the exfoliation of the CNT walls and amorphization of the remaining CNT structure. This amorphization process can be implied from the disappearance of C-C sp2 peak in the XPS spectra associated with the pi-bond network.
The impact behavior of water droplet impinging on superhydrophobic CNT arrays in a low viscosity regime is investigated for the first time. Here, the experimental data are presented in the form of several important impact behavior characteristics including critical Weber number, volume ratio, restitution coefficient, and maximum spreading diameter. As observed experimentally, three different impact regimes are identified while another impact regime is proposed. These regimes are partitioned by three critical Weber numbers, two of which are experimentally observed. The volume ratio between the primary and the secondary droplets is found to decrease with the increase of Weber number in all impact regimes other than the first one. In the first impact regime, this is found to be independent of Weber number since the droplet remains intact during and subsequent to the impingement. Experimental data show that the coefficient of restitution decreases with the increase of Weber number in all impact regimes. The rate of decrease of the coefficient of restitution in the high Weber number regime is found to be higher than that in the low and moderate Weber number. Experimental data also show that the maximum spreading factor increases with the increase of Weber number in all impact regimes. The rate of increase of the maximum spreading factor in the high Weber number regime is found to be higher than that in the low and moderate Weber number. Phenomenological approximations and interpretations of the experimental data, as well as brief comparisons to the previously proposed scaling laws, are shown here.
Dry oxidation methods are used for the first time to characterize the influence of oxidation on the capacitive behavior of CNT array EDLCs. The capacitive behavior of CNT array EDLCs can be tailored by varying their oxygen content, represented by their O/C ratio. The specific capacitance of these CNT arrays increases with the increase of their oxygen content in both KOH and Et4NBF4/PC electrolytes. As a result, their gravimetric energy density increases with the increase of their oxygen content. However, their gravimetric power density decreases with the increase of their oxygen content. The optimally oxidized CNT arrays are able to withstand more than 35,000 charge/discharge cycles in Et4NBF4/PC at a current density of 5 A/g while only losing 10% of their original capacitance.</p
Sustainable Energy Solutions for Irrigation and Harvesting in Developing Countries
Get PDFOne of the critical problems currently being faced by agriculture industry in developing nations is the alarming rate of groundwater depletion. Irrigation accounts for over 70% of the total groundwater withdrawn everyday. Compounding this issue is the use of polluting diesel generators to pump groundwater for irrigation. This has made irrigation not only the biggest consumer of groundwater but also one of the major contributors to green house gases. The aim of this thesis is to present a solution to the energy-water nexus. To make agriculture less dependent on fossil fuels, the use of a solar-powered Stirling engine as the power generator for on-farm energy needs is discussed. The Stirling cycle is revisited and practical and ideal Stirling cycles are compared. Based on agricultural needs and financial constraints faced by farmers in developing countries, the use of a Fresnel lens as a solar-concentrator and a Beta-type Stirling engine unit is suggested for sustainable power generation on the farms. To reduce the groundwater consumption and to make irrigation more sustainable, the conceptual idea of using a Stirling engine in drip irrigation is presented. To tackle the shortage of over 37 million tonnes of cold-storage in India, the idea of cost-effective solar-powered on-farm cold storage unit is discussed