La Jolla Interfaces in Science

Interdisciplinary Research Projects

Many psychological studies on naming and recognition response time are finding that subjects are slower recognizing and producing names for objects, words, and faces when the names were acquired later in life.  Surprisingly, these Age of Acquisition (AoA) effects appear independent of any frequency effects and, sometimes, stronger.  Yet, as the singular importance of frequency is deeply nestled within the core of current psychological theory, extraordinarily strong evidence will be required, now, to move it from its place.  We propose undertaking a quantitative examination of the effects of AoA and frequency at a neurobiological level in order to produce such evidence.  We have already constructed a neural network model exhibiting strong AoA effects.  With further experimentation and analysis, this model should lead us toward a theory regarding the manifestations of AoA and frequency in the neural substate--a theory we can then rigorouly verify by conducting human fIMRI studies.  return

Predoctoral Trainee: Burak Aksoylu

Co-mentors: Drs. Michael Holst and J. Andrew McCammon

Predoctoral Trainee: Nathan Baker

Co-mentors: Drs. J. Andrew McCammon and Michael Holst

Postdoctoral Trainee: Dave Barondeau

Co-mentors: Drs. John Tainer and Kevin Sullivan

Predoctoral Trainee: Kevin Briggman

Co-mentors: Drs. William B. Kristan and Henry Abarbanel

Pattern Formation and Left/Right Symmetry Breaking in Embryo Development

Postdoctoral Trainee: Javier Buceta Fernandez

Co-mentors: Drs.  Katja Lindenberg and Juan Carlos Belmonte

Using Computational Modeling for Enzyme-Ligand
Complexes and Inhibitor Design

Postdoctoral Trainee: Heather Carlson

Co-mentors: Drs. J. Andrew McCammon and Senyon Choe

Fitting Multi-Resolution Macromolecular Structures Using Fourier Correlations

New insights into cellular processes require the synthesis of information from low- to medium-resolution biophysical techniques, such as electron microscopy (EM), with atomic resolution structures.  The major aim of this project is the development of a new approach for the docking of high-resolution structures into low-resolution densities using Fourier correlation techniques.  This quantitative "internal docking" method will offer a more reliable fitting by adding surface matching terms to the volumetric correlation, in analogy to computational solutions to the "external " protein-protein docking problem.  The application of the method to 3D image reconstruction of actomyosin and kinesin-microtubule complexes from cryogenic EM will furnish a stringent test for the developed computer codes and help refine the near-atomic structure of these systems against EM data that is generated at increasingly higher resolution. return

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Exploring Quantitative Motional Models of Backbone and

Side Chain Dynamics via NMR and Computer Simulation

Postdoctoral Trainee:  Jianhan Chen

Co-mentors:  Drs. Charles Brooks and Peter Wright

Understanding the relationship between protein structure and the motional properties of the constituent atoms from measured NMR dynamic data is key to a deeper understanding of the structure-function relationship of enzymes. This project will focus on deepening this understanding through the development of more quantitative models for both backbone and side chain motions from measurements of NMR relaxation and hetero-nuclear NOE and molecular dynamics simulations. The focus of this work will be on the well-studied enzyme system of dihydrofolate reductase (DHFR), where there has been a suggestion of coupled protein dynamics and enzyme catalysis. Our efforts will be directed to a complete analysis of the motional parameters for DHFR in complex with cofactor NADPH and substrate mimic folate based on both experiment and molecular dynamics simulation. Working jointly with the groups of Professors Charles Brooks and Peter Wright at TSRI, we will examine existing NMR data from the Wright lab for side chain and backbone motional properties and perform and systematically analyze long timescale (10s of ns) dynamics trajectories of these systems. Our overall objective is the development of more quantitative models for protein motions from NMR dynamic experiments and MD simulations. return

Design and Construction of a Silicon Based Bioreactor for Hepatocytes

The current state of artificial liver design is limited by the instability of isolated hepatocytes.  In vitro, hepatocytes quickly lose key metabolic functions unless stabilized by a three-dimensional extra-cellular matrix (ECM) support.  Using existing technologies developed by the semiconductor industry as well as porous silicon chemistry techniques developed by the Sailor group, we plan to construct a silicon bioreactor that can maintain the liver specific functions of hepatocytes.  The bioreactor will contain wells coated with ECM into which the hepatocytes will be seeded.  This design provides a three-dimensional support that more closely mimics the microarchitecture of the intact liver than current therapies.  Key aspects of the bioreactor design will be driven by the Bhatia group's work on the importance of cell-cell and cell-ECM interactions in the liver.  return

 
Off-Lattice Minimalist Models to Study the Folding of Interleukin-1 Beta

We intend to focus on different levels of off-lattice models to investigate the folding of real proteins.  Off-lattice minimalist models are effective to represent global features of the energy landscape and therefore they could be useful to mimic different mechanisms of folding, that we know are utilized in nature by proteins that differ in their final folded topology.  We will be particularly interested in using experimental data as a probe of validity and efficiency of these models.  Different proteins may have different folding scenarios and may be represented by different models.  The center of our studies however will be the protein interleukin-1 beta.  The close collaboration between experiments in the Jennings group and theoretical studies in the Onuchic group should provide a full molecular understanding of the folding mechanism for this protein. return

Application of Porous Silicon Technology to the Study of

Neuronal Cell Communication and Development

The aim of the project is to use the properties of silicon (a semiconductor) and porous silicon (Sailor lab) to aid the study of interconnected neuron cell colonies (Goda lab).  The number and strength of connections between neurons (synapses) are important variables in current descriptions of thought processing and memory formation.  It has been observed that the connectivity of neurons that undergo frequent synaptic junctions and the existing neural pathways are strengthened.  The aim of this project is to incorporate the photoconductivity properties of n-type silicon to selectively stimulate identified synapses and selectively initiate intercellular communication.  The relationship between neural activity and network formation can be determined by monitoring the development of the neural cultures over time as a function of initiated synaptic communication in the early stages of neural network development. return

Structural Basis of Regional Myocardial Mechanics: In-Vivo and In-Silico Studies

Postdoctoral Trainee:  John Criscione

Co-mentors:  Drs. Andrew McCulloch and James Covell

As an LJIS trainee, I propose to work with Professors Covell and McCulloch on the mechanical role of the laminar organization of ventricular myocytes in the intact heart.  I will combine theoretical and computational modeling (in Dr. McCullochís lab) with experimental studies of three-dimensional myocardial mechanics using biplane radiography (in Dr. Covellís lab).  By altering constitutive parameters to optimize the agreement between model and experiment, the orthotropic mechanical properties of cardiac muscle will be elucidated.  In year 2, I will apply this approach to the MLP knockout mouse, which has altered laminar architecture, to study the molecular basis of orthotropic constitutive properties in the heart wall and their significance in the development of dilated cardiomyopathy. return

Mechanistic Studies of Individual Kinesin Motors

My work is focussed on understanding the molecular mechanism of movement by the biological motor protein kinesin. To accomplish this goal, I have been Using single molecule spectroscopic methods (in collaboration with Susanne Kummer and Roman Sakowicz in the laboratories of my dual mentors W.E. Moerner and L.S.B. Goldstein, respectively). Kinesin is of interest because it performs pivotal roles in several important biological processes such as mitosis, meiosis, and intracellular organelle transport as it "walks" along microtubules within cells. The mechanism of kinesin movement has been well studied in ensemble experiments, but definitive elucidation of its mechanism will require single molecule analyses of the behavior of individual moving motors. Studies performed in other laboratories have suggested a mechanism whereby kinesin walks along the microtubule in a bipedal fashion. The crystal structure, however, indicates that the accessed conformations necessary for the back foot to swing around and step 8 nm in front of the other foot would be highly strained, casting doubt on this popular mechanism. Through polarization and energy transfer experiments on single kinesin motors from a variety of organisms, we hope to further elucidate the mechanistic aspects of its motion.  return

QuantitativeAanalysis and Classification of C. elegans Behavioral Patterns

Postdoctoral Trainee: Zhaoyang (John) Feng

Co-mentors: Drs. Bill Schafer and Pamela Cosman

The nematode C. elegans has powerful genetics and a well-described nervous system, and is therefore well suited to studies of behavior at the molecular and cellular levels. However, many genes and neurons with critical roles in nervous system function have effects on nematode behavior that are subtle or difficult to describe precisely. To fully realize the potential of C. elegans as a neurobiological model will require new methods for the rapid and consistent quantitation of behavioral phenotypes. The goal of this proposed work is to develop image processing and computational modeling techniques to quantify the effects of specific genes, neurons, and pharmacological treatments on C. elegans locomotion behavior. Using these tools, we generate a database correlating molecular defects with behavioral signatures for a wide range of mutants, and develop pattern recognition applications to aid in classifying the patterns of new mutants to gain insight into their underlying molecular defect. return

The luminance (brightness) and chromaticity (color) of surfaces in natural scenes have been shown to be statistically independent.  That is, information about the luminance of a surface provides little information about the chromaticity of that surface, and vice versa.  However, I have recently shown that differencesin luminance between locations in a scene are strongly associated with differences in chromaticity.  I propose to use the statistics of these luminance and chromatic differences to construct a model that predicts whether or not two points in an image belong to the same surface.  Predicitions from this model will be compared to predictions from alternate models, and to human observers' judgments of surface boundaries.  This comparison will provide insight into how the human visual system is adapted to account for the statistics of natural scenes.  return

The efficient assembly of large molecular complexes is an integral part of the functioning of the cell.  This goal of this project is to study the assembly of the 30S subunit of the bacterial ribosome, which is a large complex of 20 proteins and a 1500+ nucleotide RNA, using a combination of microfluidics technology and state-of-the-art multi-color FRET methodologies.  Because of the size and complexity of the 30S subunit, it is very difficult to obtain detailed mechanistic information for the assembly process using conventional ensemble methods.  In the proposed approach, microfluidics will provide key capabilities for mixing of different ribosomal components in both slow and fast formats, and multi-color single molecule FRET measurements will provide the ultimate in sensitivity and resolution, providing new levels of insight into 30S assembly mechanisms.  The combined approach is also expected to be a powerful tool for study of such large biological complexes in general.   return

During my post-doctoral research in the laboratories of Joanne Chory and Joe Noel, I propose to use static and time-resolved small angle solution X-ray scattering and X-ray crystallography to determine the structures and structural dynamics that underlie the ability of phytochrome photoreceptors to convert light into biological signals.  Phytochromes are a class of photoreceptors that control a wide variety of developmental and physiological processes in plants and certain photosynthetic bacteria.  Previous studies of phytochrome have predominantly used genetic and cell biological techniques to describe the functions of the various types of phytochrome.  The proposed project allows me to apply my training in experimental biophysics and method development to a complex and biologically important sensory system and at the same time introduces me to a research environment that approaches biological questions on a cellular and organismal level. return

This project aims to investigate the dynamics of the second messenger cAMP during the development of primary neurons of Xenopus spinal cord in vitro.  We will use the FlCRhR dye developed by the Tsien lab for imaging of the activity of cAMP-dependent protein kinase by confocal microscopy.  The initial results assure us that cAMP oscillations appear to occur spontaneously in early Xenopus cells.  We will examine neurons at different stages of early development in order to determine the regulatory role of cAMP.  Kinetic modeling of this system will be very helpful to study cAMP dynamics.  return

Ligand-induced receptor proximity and clustering is a general speculation in signal transduction. However, the dynamics and mechanism have not been fully understood. In this project I investigate tumor necrosis factor receptor for its significance on cell death. The recent crystallographic evidence of tumor necrosis factor receptor inspired me to develop a lattice model to describe the clustering pattern of such receptor. Moreover, a few critical behaviors can be found from statistical mechanics. In order to understand their significance and to realize what the laws of Nature are, I am working on theoretical computation and designing highly-sensitive fluorescent experiment to explore for more evidence. return

The catalytic subunit of cAMP-dependent protein kinase (CAT) is studied as a model system of bilobal enzymes that bind substrates in a binding cleft between their two domains. We have previously studied the energy surface for transitions between closed, intermediate and open conformations of CAT by electrostatic continuum calculations on static protein conformations. Binding of ATP and a peptide inhibitor was shown to energetically stabilize the closed conformation over the open conformation.

Our present goal is to describe pathways, timescales and energy barriers of opening and closing transitions by atomistic computer simulations. What are the driving forces for these transitions? What energy barriers need to be overcome? Are these transitions slow or fast steps compared to the binding and release of substrate and product, or are they rate-limiting? In order to reduce the complexity of the system, a continuum model is being used for the solvent. The results of this analysis can be compared with data from fluorescence experiments.

A future goal might then be to extend this analysis on the inactive complex of cAMP-dependent protein kinase formed by two catalytic subunits and two regulatory subunits. How does the formation/dissociation of this complex affect the mobility of its constituents? Yet, this part of the project still awaits the resolution of a crystal structure of the holoenzyme.

The computational methods that are being developed and applied during this study will be useful for the study of protein motion in general.  return

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Dissecting the Physicochemical Principles Underlying Amyloid Peptid Aggregation

Predoctoral Trainee: Ron Hills

Co-mentors: Drs. Charles Brooks and Philip Dawson

Virtually any protein can aggregate as amyloid given the right  solution conditions, implying that amyloid behavior is a general property of the polypeptide backbone. We also know that single amino acid substitutions can have a marked effect on a protein’s amyloidogenicity. Recent studies show that chain hydrophobicity  and B-sheet propensity are often enough to predict the aggregation behavior of a given sequence, suggesting that amyloid phenomena can be explained in terms of elementary physical attributes. In order to further dissect this interplay between backbone and  sidechain interactions, we investigate the impact of introducing chemical substitutions in a model peptide. Backbone amides will be converted to esters to determine the strength of hydrogen bonds in different environments, and sidechain analogues will be employed  to study intersheet packing. The impact of these substitutions on aggregation thermodynamics and kinetics will be assessed using molecular dynamics simulations and in vitro fibril formation assays.   return

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Large Scale Classical And Ab Initio Molecular Dynamics Simulations

of Supramolecular Assemblies Involved in Flap Endonuclease-DNA Repair

Postdoctoral Trainee: Ivaylo Ivanov

Co-mentors: Drs. Andrew McCammon   and  John A. Trainer

Two general research themes will be pursued.  First, important questions related to the maintenance of economic integrity will be investigated by ab initio CPMD, classical and combined QM/MM computations.  The objective of the study is to elucidate the reactive chemistry in the active site of the DNA repair enzyme Flap Endonuclease-1 (FEN-1), while taking into account the effect of dynamic fluctuations, finite temperature effects and conformational reorganization within a realistic trajectory "steered" molecular dynamics and "computational" mutagenesis simulations to determine the protein-protein interactions responsible for binding of structurally diverse repair enzymes to Proliferating Cell Nuclear Antigen (PCNA).  We will investigate the process of binding of FEN-1 to PCNA in the context of a proposed rotary handoff mechanism for repair involving PCNA, FEN-1 and DNA Ligawse.  Both projects are aimed at answering questions of fundamental importance to chemical and molecular biology.   return

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Quantitative Rules for Plasticity and Adaptive Learning in Cortical Circuits

Predoctoral Trainee: Shantanu Jadkav

Co-mentors: Drs. Daniel Feldman  and  Terrence Sejnowski

We propose a highly integrated theoretical and experimental approach to identify the quantitative synaptic rules that mediate learning and information storage in the brain.  Synaptic learning rules describe how specific patterns of neural activity drive long-lasting changes (“plasticity”) at synapses, which is the basis for learning.   Identifying these rules will allow a quantitative, cellular-level description of learning.  To do this, we propose to record natural spike trains during training paradigms known to drive plasticity in sensory areas of cerebral cortex.  Quantitative spike train analysis techniques will be developed and used to identify spike train features that may drive plasticity.  In vitro synaptic physiology experiments will test whether these features actually cause plasticity, and will characterize the relevant learning rules.  We will build a realistic computational model of cortex implementing these rules, to test whether they explain known features of cortical plasticity and information storage.   return

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Probing the Folding Thermodynamics and Kinetics of a WW Domain by

Single Molecule Fluorescence Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS)

Postdoctoral Trainee: Marcus Jäger

Co-mentors: Drs. Jeffery Kelly and David Millar

The folding thermodynamics of a WW-domain will be investigated at single molecule resolution. Fluorescence donor/acceptor pairs will be incorporated into the WW domain by peptide synthesis. Equilibrium unfolding curves will be obtained using FRET distance measurements as an effective reaction coordinate. Using FRET histograms, the folded and unfolded states can be directly observed. The fractional occupation of the native state can be calculated by peak integration. A plot of this fraction vs. denaturant can be constructed and compared with similar plots from the ensemble regime. A coincidence of the two plots will confirm a two-state unfolding model. Non-superimposable plots imply a multistep process involving intermediates, that are hidden in traditional ensemble measurements.

The folding dynamics is studied at equilibrium in the presence of increasing concentrations of chaotrope under confocal configuration. FRET intensities will be recorded as a function of time and the second order correlation function will be calculated. The microscopic rate constants for (un)folding will be extracted from the autocorrelation function by deconvolution. Using site-directed mutants, the transition state for folding will be mapped.

The overall goal of this work is to establish the existence and chemical identity of novel vertebrate hormones.   The nuclear receptors are a large superfamily of ligand-activated transcription factors that influence transcription either directly, through specific binding to the regulatory regions of target genes, or indirectly, via protein-protein interactions with other transcription factors. This superfamily of ligand-dependent transcription factors includes receptors for a variety of small, lipid-soluble hormones including cortisol, aldosterone, estrogen, progesterone, testosterone, vitamin D3, thyroid hormone and retinoic acid. The term orphan receptor was coined in the late 1980s to describe the first of what has become a large number of novel gene products that by homology belong to the nuclear receptor superfamily, but for which ligands are initially unknown. A major challenge is to decipher the function of these proteins, to determine whether they are hormone responsive, and, if so, to identify their cognate ligands. An initial understanding of orphan receptor function is established through comparative studies of sequence identity, DNA-binding, dimerization, and ligand-independent activities. The major strategy for identifying orphan receptor ligands has been the use of the cotransfection screening assay in which the orphan activates a reporter plasmid in the presence of a cognate ligand. Using this strategy, at least six lipophilic hormone-like activators have been discovered for orphan receptors. Previous studies have relied on the ability to obtain candidate orphan ligands from commercially available sources. This approach relies on a hit or miss strategy and thus depends on a large number of available compounds. Besides its obvious expense, the identified ligands are not assumed to exist in natural tissues or fluids. Accordingly, my goal is to use these materials as a starting source to actually extract and fractionate bona-fide ligands.

My approach to identification of ligands for nuclear orphan receptors requires combined skills of molecular biology and organic chemistry including isolation and structural characterization of small organic molecules. I believe that the combination of natural product chemistry and receptor biology is both a unique and fruitful approach to biological problems. I also believe that this approach promises a way to identify natural regulators and thus to understand the cause and mechanism underlying physiology and human diseases.  return

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Exploring B-Sheet Formation

Predoctoral Trainee:  John Karanicolas

Co-mentors: Drs.  Charles Brooks III and Jeffery Kelly

In order to achieve functional activity, most proteins must first fold to a specific "native" conformation from a wide array of unfolded conformations.  The mechanism of B-Sheet formation is of particular interest, due to its implication in protein misfolding and misassembly diseases such as bovine spongiform encephalopathy.  In order to deepen our understanding of B-Sheet formation, we turn our attention to the Pin WW domain, a small three-stranded antiparallel sheet.  Variants of this protein have been the subject of thermodynamic and kinetic characterization in the Kelly lab, in order to determine the contribution of individual residues to stability and folding.  These results are in agreement with preliminary results obtained theoretically in the Brooks lab using a simplified model.  The objective of the research described herein is to use both this simplified representation as well as an all-atom representation in order to both understand and interpret the results of these experiments and propose further experiments to be carried out in the Kelly lab.   return

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Single Molecule Studies of Chromatin Assembly using Optical Tweezers

Postdoctoral Trainee: Pu-Chun Ke

Co-mentors: Drs. Douglas Smith and James Kadonaga

I propose to use optical tweezers to carry out single molecule studies of chromatin assembly catalyzed by ACF, an ATP dependent assembly factor that is believed to act as a molecular motor. The aim of these studies is to provide an in-depth understanding of the biophysical mechanism of chromatin assembly, a problem of vital importance in molecular and cell biology. Optical tweezers will allow us to directly measure the interaction of ACF with single DNA molecules and to directly probe the DNA compaction forces generated by ACF in real time. The goals of this project include designing a custom-made optical tweezer system, learning and adapting the in-vitro system for chromatin assembly, and characterizing the motor properties of ACF. This research will be a collaborative effort between Prof. Smith's lab in the Department of Physics and Prof. Kadonaga's lab in the Department of Biology at UCSD. return

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Mechanical Study of Histone Modified Chromatin Using Optical Tweezers

Predoctoral Trainee:  Angela Klohs-Foudray

Co-mentors: Drs.  Douglas Smith and James Kadonaga

The past decade has seen great advances in the understanding of the role of chromatin structure in transcriptional activitiy, cellular identity, and fate.  Techniques such as single DNA molecule manipulation have revealed even greater depth of understanding by probing individual structures, though many questions still remain.  I propose to use optical tweezers to determine if histone modification leads to a change in the mechanical properties of the structure of chromatin.  The effect of induced alteration will be observed on chromatin with a predetermined, static in-vitro modification as well as on dynamically modified chromatin.  The goals of this project will include the design, construction and implementation of an optical tweezers system in Professor Doug Smith's lab in the Physics Department; purification and modification of chromatin in Professor Kadonaga's lab in the Biology Department; and characterization of the mechanical properties of chromatin.

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Fast Rotational and Flexible Docking

Postdoctoral Trainee: Julio Kovacs

Co-mentors: Drs. Willy Wriggers and Mike Holst

The major aim of this project is to develop new, computationally efficient methods for both rigid-body and flexible docking of macromolecular structures over different levels of resolution. The rigid-body part uses a new parametrization of the 3D rotation group, which, in combination with Fourier techniques, allows to compute the "rotational correlation function" quickly. The rigid-body fitting thus obtained is then used as the starting step for the flexible docking part, which provides not only the final correspondence of the given structures, but the full deformation path between them.

These methods will contribute to a better understanding of the dynamics and conformational changes of macromolecular assemblies. In addition, a slight variant of the above algorithms will be useful for the "exterior docking" problem, of great importance in drug design. return

One of the goals of studying protein-protein interactions is to understand how two or more proteins preferentially associate with one another to form a complex. Determining the structure of a complex by traditional methods such as NMR or X-ray crystallography is extraordinarily time consuming. This research proposal describes methodology to be developed that will enable the rapid determination of protein-protein interactions by two unrelated but complimentary techniques. Mass spectrometry will be used to monitor the rate of proton-deuteron exchange in the protein complex vs. the free proteins in order to determine what parts of the surface are at the interface. Computational methods will be used to rapidly evaluate the electrostatic potential energy of interaction between the two molecules. These energies are then used to determine the most favorable sites of interaction. This calculation is carried out by using mathematical convolutions on a highly parallel supercomputer.

With the present state of the art, neither approach alone is completely conclusive because each produces a list of possible interactions. The final stage of work on this project will be to integrate the data from both techniques in order to substantially narrow the list of possibilities to a useful number.  return

Third harmonics of ultrashort laser pulses are produced in a tight focus if optical conditions change within the focal volume; if the focus lies in a region of uniform optical properties, third harmonic production is strongly inhibited.  It is therefore possible to produce optically sectioned images of the surfaces of microscopic bodies, analogous to those produced by multi-photon microscopy or confocal microscopy.  Furthermore, since third harmonic generation is sensitive to changes in local optical conditions and since the optical properties of neurons change according to electrochemical state, the production of third harmonic light will be affected by changes in the trans-membrane potential of neurons, providing a non-invasive technique with which to image neuronal activity.  This project will investigate this modulation of third harmonic generation at a tight focus, proceeding from molecular studies of voltage sensitivity to the goal of making quantitative measurements of neurons as they fire. return

We will combine the DOT and CGU algorithms developed independently by co-mentors Rosen, Ten Eyck and their colleagues.  The DOT algorithm computes the potential energy associated to a pair of rigid macromolecules using convolution integrals and Fast Fourier Transforms (FFTs).  CGU determines the global minimum of an energy function using an iterated algorithm which closely underestimates large numbers of global minima with a convex function.  The CGU-DOT combined algorithm will be used to rapidly compute and minimize energy functions that govern the interactions between macromolecules.  In order to employ the combined algorithm most efficiently, it will be necessary to control the regularity of potential energies computed with DOT.  The control of regularity will be examined in light of recent developments between differentiability and fractional dimension.  In particular, it is felt this will allow complex macromolecules to be analyzed using computationally efficient means.

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Theoretical and Experimental Exploration of

Inter-Protein Electron Transfer Reactions

Postdoctoral Trainee: Osamu Miyashita

Co-mentors: Drs. José Onuchic and Mel Okamura

Inter-protein electron transfer (ET) plays a central role in bioenergetics, drug metabolism and DNA damage. A typical system for these reactions is the ET process between cytochrome c2 and the photosynthetic reaction center (PRC). Our goal is to understand how the dynamics of cytochrome c2 docking onto the PRC and specific docked configurations mediate the observed ET rate. There are two possible interpretations for the experimental evidence that the second-order ET rate is correlated with the binding-free-energy with a Bronsted coefficient of 0.4. First, the activation barrier is overcome after 40% of the complex stabilization is achieved, and complex formation is fast beyond this stage. The other possibility is that the first-order rate may be sufficiently fast for pre-complex configurations. In such a case, ET would take place before the full stable complex is formed. We will determine which of these possibilities, or an intermediate situation between them, controls the ET mechanism.    return

My project will deal with understanding various aspects of the life cycle of a small, icosahedral insect RNA virus called Flock House Virus. Due to its simplicity, both genomic and structural, FHV makes an excellent model system within which to study such basic life cycle components as viral assembly, maturation and host cell invasion. These events allow the virus to reproduce and propagate itself throughout a host cell population and are thus important targets for investigation. My proposed project will attempt to answer the following questions:
 
1. What are the energetic factors involved in the assembly process for FHV?
2. What energetic and/or structural role do both surface and internal calcium ions play in the ability of the virus particle to assemble as well as in its advancement from provirion to mature, infectious particle during the self-catalyzed cleavage event?
3. Can the putative transmembrane RNA-trafficking function of the 5-fold amphipathic helical bundle released upon maturation be inferred from and modeled by molecular dynamics simulations?
These questions, and others that will arise as we study the energetics of various aspects of the FHV life cycle, will aide in understanding these fascinating biological entities with the goal that basic principles of icosahedral virus particle design and dynamics will be elucidated which will take us beyond the static picture we have at the present. To this