Design, synthesis and characterization of sequence-specific DNA-cleaving metalloproteins

This thesis describes research pursued in two areas, both involving the design and synthesis of sequence specific DNA-cleaving proteins. The first involves the use of sequence-specific DNA-cleaving metalloproteins to probe the structure of a protein-DNA complex, and the second seeks to develop cleaving moieties capable of DNA cleavage through the generation of a non-diffusible oxidant under physiological conditions.

Chapter One provides a brief review of the literature concerning sequence-specific DNA-binding proteins. Chapter Two summarizes the results of affinity cleaving experiments using leucine zipper-basic region (bZip) DNA-binding proteins. Specifically, the NH_2-terminal locations of a dimer containing the DNA binding domain of the yeast transcriptional activator GCN4 were mapped on the binding sites 5'-CTGACTAAT-3' and 5'ATGACTCTT- 3' using affinity cleaving. Analysis of the DNA cleavage patterns from Fe•EDTA-GCN4(222-281) and (226-281) dimers reveals that the NH_2-termini are in the major groove nine to ten base pairs apart and symmetrically displaced four to five base pairs from the central C of the recognition site. These data are consistent with structural models put forward for this class of DNA binding proteins. The results of these experiments are evaluated in light of the recently published crystal structure for the GCN4-DNA complex. Preliminary investigations of affinity cleaving proteins based on the DNA-binding domains of the bZip proteins Jun and Fos are also described.

Chapter Three describes experiments demonstrating the simultaneous binding of GCN4(226-281) and 1-Methylimidazole-2-carboxamide-netropsin (2-ImN), a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an antiparallel, side-by-side dimer. Through the use of Fe•EDTA-GCN4(226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4(226-281) and the dimeric peptide 2- ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively. The association constants for 2-ImN in the presence and in the absence of Fe•EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.

Chapter Four describes the synthesis and characterization of PBA-β-OH-His- Hin(139-190), a hybrid protein containing the DNA-binding domain of Hin recombinase and the putative iron-binding and oxygen-activating domain of the antitumor antibiotic bleomycin. This 54-residue protein, comprising residues 139-190 of Hin recombinase with the dipeptide pyrimidoblamic acid-β-hydroxy-L-histidine (PBA-β-OH-His) at the NH2 terminus, was synthesized by solid phase methods. PBA-β-OH-His-Hin(139- 190) binds specifically to DNA at four distinct Hin binding sites with affinities comparable to those of the unmodified Hin(139-190). In the presence of dithiothreitol (DTT), Fe•PB-β-OH-His-Hin(139-190) cleaves DNA with specificity remarkably similar to that of Fe•EDTA-Hin(139-190), although with lower efficiency. Analysis of the cleavage pattern suggests that DNA cleavage is mediated through a diffusible species, in contrast with cleavage by bleomycin, which occurs through a non-diffusible oxidant.

A bayesian probabilistic approach to structural health monitoring

A Bayesian probabilistic methodology for on-line structural health monitoring which addresses the issue of parameter uncertainty inherent in problem is presented. The method uses modal parameters for a limited number of modes identified from measurements taken at a restricted number of degrees of freedom of a structure as the measured structural data. The application presented uses a linear structural model whose stiffness matrix is parameterized to develop a class of possible models. Within the Bayesian framework, a joint probability density function (PDF) for the model stiffness parameters given the measured modal data is determined. Using this PDF, the marginal PDF of the stiffness parameter for each substructure given the data can be calculated.

Monitoring the health of a structure using these marginal PDFs involves two steps. First, the marginal PDF for each model parameter given modal data from the undamaged structure is found. The structure is then periodically monitored and updated marginal PDFs are determined. A measure of the difference between the calibrated and current marginal PDFs is used as a means to characterize the health of the structure. A procedure for interpreting the measure for use by an expert system in on-line monitoring is also introduced.

The probabilistic framework is developed in order to address the model parameter uncertainty issue inherent in the health monitoring problem. To illustrate this issue, consider a very simplified deterministic structural health monitoring method. In such an approach, the model parameters which minimize an error measure between the measured and model modal values would be used as the "best" model of the structure. Changes between the model parameters identified using modal data from the undamaged structure and subsequent modal data would be used to find the existence, location and degree of damage. Due to measurement noise, limited modal information, and model error, the "best" model parameters might vary from one modal dataset to the next without any damage present in the structure. Thus, difficulties would arise in separating normal variations in the identified model parameters based on limitations of the identification method and variations due to true change in the structure. The Bayesian framework described in this work provides a means to handle this parametric uncertainty.

The probabilistic health monitoring method is applied to simulated data and laboratory data. The results of these tests are presented.

A biochemical and structural characterization of Drosophila neuroglian

A number of cell-cell interactions in the nervous system are mediated by immunoglobulin gene superfamily members. For example, neuroglian, a homophilic neural cell adhesion molecule in Drosophila, has an extracellular portion comprising six C- 2 type immunoglobulin-like domains followed by five fibronectin type III (FnIII) repeats. Neuroglian shares this domain organization and significant sequence identity with Ll, a murine neural adhesion molecule that could be a functional homologue. Here I report the crystal structure of a proteolytic fragment containing the first two FnIII repeats of neuroglian (NgFn 1,2) at 2.0Å. The interpretation of photomicrographs of rotary shadowed Ng, the entire extracellular portion of neuroglian, and NgFnl-5, the five neuroglian Fn III domains, is also discussed.

The structure of NgFn 1,2 consists of two roughly cylindrical β-barrel structural motifs arranged in a head-to-tail fashion with the domains meeting at an angle of ~120, as defined by the cylinder axes. The folding topology of each domain is identical to that previously observed for single FnIII domains from tenascin and fibronectin. The domains of NgFn1,2 are related by an approximate two fold screw axis that is nearly parallel to the longest dimension of the fragment. Assuming this relative orientation is a general property of tandem FnIII repeats, the multiple tandem FnIII domains in neuroglian and other proteins are modeled as thin straight rods with two domain zig-zag repeats. When combined with the dimensions of pairs of tandem immunoglobulin-like domains from CD4 and CD2, this model suggests that neuroglian is a long narrow molecule (20 - 30 Å in diameter) that extends up to 370Å from the cell surface.

In photomicrographs, rotary shadowed Ng and NgFn1-5 appear to be highly flexible rod-like molecules. NgFn 1-5 is observed to bend in at least two positions and has a mean total length consistent with models generated from the NgFn1,2 structure. Ng molecules have up to four bends and a mean total length of 392 Å, consistent with a head-to-tail packing of neuroglian's C2-type domains.

Modeling and analysis of hysteretic structural behavior

For damaging response, the force-displacement relationship of a structure is highly nonlinear and history-dependent. For satisfactory analysis of such behavior, it is important to be able to characterize and to model the phenomenon of hysteresis accurately. A number of models have been proposed for response studies of hysteretic structures, some of which are examined in detail in this thesis. There are two popular classes of models used in the analysis of curvilinear hysteretic systems. The first is of the distributed element or assemblage type, which models the physical behavior of the system by using well-known building blocks. The second class of models is of the differential equation type, which is based on the introduction of an extra variable to describe the history dependence of the system.

Owing to their mathematical simplicity, the latter models have been used extensively for various applications in structural dynamics, most notably in the estimation of the response statistics of hysteretic systems subjected to stochastic excitation. But the fundamental characteristics of these models are still not clearly understood. A response analysis of systems using both the Distributed Element model and the differential equation model when subjected to a variety of quasi-static and dynamic loading conditions leads to the following conclusion: Caution must be exercised when employing the models belonging to the second class in structural response studies as they can produce misleading results.

The Massing's hypothesis, originally proposed for steady-state loading, can be extended to general transient loading as well, leading to considerable simplification in the analysis of the Distributed Element models. A simple, nonparametric identification technique is also outlined, by means of which an optimal model representation involving one additional state variable is determined for hysteretic systems.

Models of the Oxygen-Evolving Complex of Photosystem II

In the five chapters that follow, I delineate my efforts over the last five years to synthesize structurally and chemically relevant models of the Oxygen Evolving Complex (OEC) of Photosystem II. The OEC is nature’s only water oxidation catalyst, in that it forms the dioxygen in our atmosphere necessary for oxygenic life. Therefore understanding its structure and function is of deep fundamental interest and could provide design elements for artificial photosynthesis and manmade water oxidation catalysts. Synthetic endeavors towards OEC mimics have been an active area of research since the mid 1970s and have mutually evolved alongside biochemical and spectroscopic studies, affording ever-refined proposals for the structure of the OEC and the mechanism of water oxidation. This research has culminated in the most recent proposal: a low symmetry Mn₄CaO₅ cluster with a distorted Mn₃CaO₄ cubane bridged to a fourth, dangling Mn. To give context for how my graduate work fits into this rich history of OEC research, Chapter 1 provides a historical timeline of proposals for OEC structure, emphasizing the role that synthetic Mn and MnCa clusters have played, and ending with our Mn₃CaO₄ heterometallic cubane complexes.

In Chapter 2, the triarylbenzene ligand framework used throughout my work is introduced, and trinuclear clusters of Mn, Co, and Ni are discussed. The ligand scaffold consistently coordinates three metals in close proximity while leaving coordination sites open for further modification through ancillary ligand binding. The ligands coordinated could be varied, with a range of carboxylates and some less coordinating anions studied. These complexes’ structures, magnetic behavior, and redox properties are discussed.

Chapter 3 explores the redox chemistry of the trimanganese system more thoroughly in the presence of a fourth Mn equivalent, finding a range of oxidation states and oxide incorporation dependent on oxidant, solvent, and Mn salt. Oxidation states from Mn^II₄ to Mn^IIIMn^IV₃ were observed, with 1-4 O^2– ligands incorporated, modeling the photoactivation of the OEC. These complexes were studied by X-ray diffraction, EPR, XAS, magnetometry, and CV.

As Ca²⁺ is a necessary component of the OEC, Chapter 4 discusses synthetic strategies for making highly structurally accurate models of the OEC containing both Mn and Ca in the Mn₃CaO₄ cubane + dangling Mn geometry. Structural and electrochemical characterization of the first Mn₃CaO₄ heterometallic cubane complex— and comparison to an all-Mn Mn₄O₄ analog—suggests a role for Ca²⁺ in the OEC. Modification of the Mn₃CaO₄ system by ligand substitution affords low symmetry Mn₃CaO₄ complexes that are the most accurate models of the OEC to date.

Finally, in Chapter 5 the reactivity of the Mn₃CaO₄ cubane complexes toward O- atom transfer is discussed. The metal M strongly affects the reactivity. The mechanisms of O-atom transfer and water incorporation from and into Mn₄O₄ and Mn₄O₃ clusters, respectively, are studied through computation and ¹⁸O-labeling studies. The μ3-oxos of the Mn₄O₄ system prove fluxional, lending support for proposals of O^2– fluxionality within the OEC.

Exploring the Kinetics of Domain Switching in Ferroelectrics for Structural Applications

The complex domain structure in ferroelectrics gives rise to electromechanical coupling, and its evolution (via domain switching) results in a time-dependent (i.e. viscoelastic) response. Although ferroelectrics are used in many technological applications, most do not attempt to exploit the viscoelastic response of ferroelectrics, mainly due to a lack of understanding and accurate models for their description and prediction. Thus, the aim of this thesis research is to gain better understanding of the influence of domain evolution in ferroelectrics on their dynamic mechanical response. There have been few studies on the viscoelastic properties of ferroelectrics, mainly due to a lack of experimental methods. Therefore, an apparatus and method called Broadband Electromechanical Spectroscopy (BES) was designed and built. BES allows for the simultaneous application of dynamic mechanical and electrical loading in a vacuum environment. Using BES, the dynamic stiffness and loss tangent in bending and torsion of a particular ferroelectric, viz. lead zirconate titanate (PZT), was characterized for different combinations of electrical and mechanical loading frequencies throughout the entire electric displacement hysteresis. Experimental results showed significant increases in loss tangent (by nearly an order of magnitude) and compliance during domain switching, which shows promise as a new approach to structural damping. A continuum model of the viscoelasticity of ferroelectrics was developed, which incorporates microstructural evolution via internal variables and associated kinetic relations. For the first time, through a new linearization process, the incremental dynamic stiffness and loss tangent of materials were computed throughout the entire electric displacement hysteresis for different combinations of mechanical and electrical loading frequencies. The model accurately captured experimental results. Using the understanding gained from the characterization and modeling of PZT, two applications of domain switching kinetics were explored by using Micro Fiber Composites (MFCs). Proofs of concept of set-and-hold actuation and structural damping using MFCs were demonstrated.

Quantifying Earthquake Collapse Risk of Tall Steel Moment Frame Buildings Using Rupture-to-Rafters Simulations

There is a sparse number of credible source models available from large-magnitude past earthquakes. A stochastic source model generation algorithm thus becomes necessary for robust risk quantification using scenario earthquakes. We present an algorithm that combines the physics of fault ruptures as imaged in laboratory earthquakes with stress estimates on the fault constrained by field observations to generate stochastic source models for large-magnitude (Mw 6.0-8.0) strike-slip earthquakes. The algorithm is validated through a statistical comparison of synthetic ground motion histories from a stochastically generated source model for a magnitude 7.90 earthquake and a kinematic finite-source inversion of an equivalent magnitude past earthquake on a geometrically similar fault. The synthetic dataset comprises of three-component ground motion waveforms, computed at 636 sites in southern California, for ten hypothetical rupture scenarios (five hypocenters, each with two rupture directions) on the southern San Andreas fault. A similar validation exercise is conducted for a magnitude 6.0 earthquake, the lower magnitude limit for the algorithm. Additionally, ground motions from the Mw7.9 earthquake simulations are compared against predictions by the Campbell-Bozorgnia NGA relation as well as the ShakeOut scenario earthquake. The algorithm is then applied to generate fifty source models for a hypothetical magnitude 7.9 earthquake originating at Parkfield, with rupture propagating from north to south (towards Wrightwood), similar to the 1857 Fort Tejon earthquake. Using the spectral element method, three-component ground motion waveforms are computed in the Los Angeles basin for each scenario earthquake and the sensitivity of ground shaking intensity to seismic source parameters (such as the percentage of asperity area relative to the fault area, rupture speed, and risetime) is studied.

Under plausible San Andreas fault earthquakes in the next 30 years, modeled using the stochastic source algorithm, the performance of two 18-story steel moment frame buildings (UBC 1982 and 1997 designs) in southern California is quantified. The approach integrates rupture-to-rafters simulations into the PEER performance based earthquake engineering (PBEE) framework. Using stochastic sources and computational seismic wave propagation, three-component ground motion histories at 636 sites in southern California are generated for sixty scenario earthquakes on the San Andreas fault. The ruptures, with moment magnitudes in the range of 6.0-8.0, are assumed to occur at five locations on the southern section of the fault. Two unilateral rupture propagation directions are considered. The 30-year probabilities of all plausible ruptures in this magnitude range and in that section of the fault, as forecast by the United States Geological Survey, are distributed among these 60 earthquakes based on proximity and moment release. The response of the two 18-story buildings hypothetically located at each of the 636 sites under 3-component shaking from all 60 events is computed using 3-D nonlinear time-history analysis. Using these results, the probability of the structural response exceeding Immediate Occupancy (IO), Life-Safety (LS), and Collapse Prevention (CP) performance levels under San Andreas fault earthquakes over the next thirty years is evaluated.

Furthermore, the conditional and marginal probability distributions of peak ground velocity (PGV) and displacement (PGD) in Los Angeles and surrounding basins due to earthquakes occurring primarily on the mid-section of southern San Andreas fault are determined using Bayesian model class identification. Simulated ground motions at sites within 55-75km from the source from a suite of 60 earthquakes (Mw 6.0 − 8.0) primarily rupturing mid-section of San Andreas fault are considered for PGV and PGD data.

SteelConverter and Caltech VirtualShaker: Rapid Nonlinear Cloud-Based Structural Model Conversion and Analysis

STEEL, the Caltech created nonlinear large displacement analysis software, is currently used by a large number of researchers at Caltech. However, due to its complexity, lack of visualization tools (such as pre- and post-processing capabilities) rapid creation and analysis of models using this software was difficult. SteelConverter was created as a means to facilitate model creation through the use of the industry standard finite element solver ETABS. This software allows users to create models in ETABS and intelligently convert model information such as geometry, loading, releases, fixity, etc., into a format that STEEL understands. Models that would take several days to create and verify now take several hours or less. The productivity of the researcher as well as the level of confidence in the model being analyzed is greatly increased.

It has always been a major goal of Caltech to spread the knowledge created here to other universities. However, due to the complexity of STEEL it was difficult for researchers or engineers from other universities to conduct analyses. While SteelConverter did help researchers at Caltech improve their research, sending SteelConverter and its documentation to other universities was less than ideal. Issues of version control, individual computer requirements, and the difficulty of releasing updates made a more centralized solution preferred. This is where the idea for Caltech VirtualShaker was born. Through the creation of a centralized website where users could log in, submit, analyze, and process models in the cloud, all of the major concerns associated with the utilization of SteelConverter were eliminated. Caltech VirtualShaker allows users to create profiles where defaults associated with their most commonly run models are saved, and allows them to submit multiple jobs to an online virtual server to be analyzed and post-processed. The creation of this website not only allowed for more rapid distribution of this tool, but also created a means for engineers and researchers with no access to powerful computer clusters to run computationally intensive analyses without the excessive cost of building and maintaining a computer cluster.

In order to increase confidence in the use of STEEL as an analysis system, as well as verify the conversion tools, a series of comparisons were done between STEEL and ETABS. Six models of increasing complexity, ranging from a cantilever column to a twenty-story moment frame, were analyzed to determine the ability of STEEL to accurately calculate basic model properties such as elastic stiffness and damping through a free vibration analysis as well as more complex structural properties such as overall structural capacity through a pushover analysis. These analyses showed a very strong agreement between the two softwares on every aspect of each analysis. However, these analyses also showed the ability of the STEEL analysis algorithm to converge at significantly larger drifts than ETABS when using the more computationally expensive and structurally realistic fiber hinges. Following the ETABS analysis, it was decided to repeat the comparisons in a software more capable of conducting highly nonlinear analysis, called Perform. These analyses again showed a very strong agreement between the two softwares in every aspect of each analysis through instability. However, due to some limitations in Perform, free vibration analyses for the three story one bay chevron brace frame, two bay chevron brace frame, and twenty story moment frame could not be conducted. With the current trend towards ultimate capacity analysis, the ability to use fiber based models allows engineers to gain a better understanding of a building’s behavior under these extreme load scenarios.

Following this, a final study was done on Hall’s U20 structure [1] where the structure was analyzed in all three softwares and their results compared. The pushover curves from each software were compared and the differences caused by variations in software implementation explained. From this, conclusions can be drawn on the effectiveness of each analysis tool when attempting to analyze structures through the point of geometric instability. The analyses show that while ETABS was capable of accurately determining the elastic stiffness of the model, following the onset of inelastic behavior the analysis tool failed to converge. However, for the small number of time steps the ETABS analysis was converging, its results exactly matched those of STEEL, leading to the conclusion that ETABS is not an appropriate analysis package for analyzing a structure through the point of collapse when using fiber elements throughout the model. The analyses also showed that while Perform was capable of calculating the response of the structure accurately, restrictions in the material model resulted in a pushover curve that did not match that of STEEL exactly, particularly post collapse. However, such problems could be alleviated by choosing a more simplistic material model.