Neurodegenerative disorders, including prion diseases, are associated with protein aggregation. Considerable evidence indicates that the extent of neurotoxicity or infectivity of the oligomeric or aggregated proteins is strictly dependent on their structures. The proteins involved in these diseases are conformationally heterogeneous and, according to an emerging view, can switch back-and-forth between functional and various amyloidogenic/prionogenic conformations (strain conformations). Since the various conformations present in these heterogeneous ensembles lead to different aggregated forms, characterizing the monomer conformational space is critical to understand their pathogenecity. However, the characterization of the conformational heterogeneity of monomeric proteins by standard structural biological methods has proven to be extremely challenging. Single-molecule methodologies offer exciting opportunities to increase our understanding of protein conformational equilibrium. This project is aimed at using a novel approach that combines the two main methods for single-molecule manipulation, Optical Tweezers and Atomic Force Microscopy, to map the conformational energy landscapes of a mammalian prion protein (PrP) and yeast prion Sup35 protein, as well as their monomer conformations that promote the aggregation process. Mouse PrP is an ideal candidate for prion conformational studies due to the possibility of creating different infectious conformations in a test tube and of introducing them in mice to develop novel prion diseases. Similarly, the ability of Sup35NM, a prion domain of Sup35, to convert into prion states in a test tube and the possibility of introducing them in yeast, make the yeast prion [PSI+] system uniquely suited to investigate the mechanism by which a single polypeptide (Sup35NM), displaying a heterogeneous ensemble of conformations, can form initial nuclei and misfolds into self-propagating distinct amyloid conformations. The synergic integration of these single-molecule techniques will make it possible to explore conformational properties of both mammalian PrP and yeast prion that are inaccessible to more classical in-bulk ensemble-averaged methods, and to study the nature of the energy surfaces over which these molecules diffuse as they move between their different strain conformations and towards those that are responsible for triggering the aggregation processes and pathogenesis.

Ubiquitination is used by all eukaryotic cells to regulate the localization and turn-over of membrane proteins. Ubiquitination is mediated by ubiquitin ligases. This proposal brings together four researchers working in three continents to characterize a family of such ligases called MARCHs. The MARCHs play major immunoregulatory roles and are anchored to membranes, a unique feature among ubiquitin ligases. Indeed, they recognize their substrates via transmembrane (TM) region interactions, itself a poorly understood phenomenon. We will apply our expertise in immunology, cell biology, proteomics, solution NMR and computation biophysics to fulfill the following aims:

AIM 1: To identify MARCHs substrates

Aim 1.1: We will characterize the expression pattern of MARCH genes in the immune system.

Aim 1.2: We will use label-free quantitative proteomics of plasma membrane (PM) fractions to identify proteins with altered surface expression levels in cells deficient in MARCH 1, 2, 3, 4, 8 or 9 (MARCH substrates).

AIM 2: To characterize the structural basis of MARCH substrate recognition via TM interactions

Aim 2.1: We will determine the role of structural motifs in the TM regions of the MARCHs and their substrates in MARCH-substrate recognition.

Aim 2.2: We will characterize the three-dimensional arrangements of TM regions of the MARCHs and their substrates using solution NMR.

Aim 2.3: We will apply computational modeling to the study of interactions between the TM regions of the MARCHs and their substrates.

Aim 2.4: We will generate chimeric mice with bone marrow retrovirally transduced to express mutant forms of the MARCHs to validate the conclusions of the structural studies in vivo.

The knowledge we will gain with this project will be highly significant at multiple levels. It will contribute to understanding the structural basis of TM-mediated protein-protein recognition and the role of ubiquitination in regulation of membrane protein expression and localization, an activity present in all eukaryotic cells. As the MARCH substrates that we will identify are likely involved in immunoregulation, this project will also be relevant to immunological tolerance and the initiation of immunity. Finally, our project will lead to refinement of strategies for computational modeling of three-dimensional structures of membrane proteins.

Neuronal morphology and function relies on microtubules for transport of proteins and membranes; impairment of these transport functions may contribute to neurodegenerative disease. Currently, little is known about microtubule dynamics in the axon. The prevailing view is that all axonal microtubules are produced by the centrosome and then transported down the axon as stable polymers. However, very recently, it was shown that axons grow in the absence of centrosomes. Thus, acentrosomal microtubule nucleation may have a major role in axon extension, although the mechanism is unknown. Two missing tools have hindered our understanding of axonal microtubules: visualization of individual microtubule dynamics in axons, and molecular tools for understanding microtubule nucleation in acentrosomal arrays. My first aim will be to apply modern light microscopy methods to visualize microtubule dynamics using rat hippocampal cells and the squid giant axon. My second aim will be to further characterize the microtubule landscape in the axon in terms of stability and post-translational modifications and understand these modifications in the context of microtubule dynamics, transport and function. My third aim will be to identify the roles of two new microtubule interacting proteins - Augmin and Patronin in neurons. Molecular reagents for Augmin-g-TURC complex may provide new information about microtubule nucleation along the axon. In addition, I will also explore the role of Patronin a protein involved in capping microtubule minus-end. These new imaging and molecular assays should provide the first details about the dynamics and properties of axonal microtubules.

All phenomena of the living world shape, and are shaped by, the process of evolution. In perhaps no other area of biology this fundamental duality becomes as evident as in the complex communication systems that mediate mate recognition in the animal kingdom. Both neurobiologically and evolutionarily, the task of recognizing a potential mate for reproduction is truly a multiple-task including the fundamental challenge of sex and species recognition as well as the more sophisticated exercise of taxing the potential mate’s fitness, i.e. its likely reproductive value. Unsurprisingly thus, when it comes to their mating decisions animals usually rely on signals from multiple sensory information channels. One sensory system that is frequently involved in the mating negotiations of both vertebrates and invertebrates is the sense of hearing: From marine mammals to air-borne mosquitoes, acoustic courtship rituals have evolved that include the generation, reception and subsequent analysis of species-specific sound signals. A prominent example is the acoustic courtship of flies of the genus Drosophila. As part of their mating ritual, male flies sing a ‘love song’ to their females. These songs differ across fly species and are deemed to contribute to both species isolation and speciation in Drosophilid flies. We here attempt to elucidate the evolutionary, material and mechanistic bases of species-recognition in the Drosophila acoustic communication system. Applying state-of-the art techniques in biophysics, bioinformatics, genetics, bioengineering, neuroanatomy, behavioural analysis, and in vivo electrophysiology, across the 12 recently sequenced Drosophila species, we will explicitly probe for species-specific mechanisms underlying the emission, reception, and processing of sound in the fly. Our project represents an unprecedented multi-level approach, not only to acoustic courtship in Drosophila, but to animal communication, molecular evolution and speciation in general.

Faithful chromosome segregation is essential for the proliferation of all organisms. Although studies in model eukaryotic organisms have found that the kinetochore complex formed on centromeres facilitates their chromosome segregation, it is not known if this mechanism applies to all eukaryotes. To uncover novel principles of the molecular machinery that drives segregation, I will study chromosome segregation in Trypanosoma brucei because this protozoan parasite branched early in eukaryotic evolution and has various unusual features. Trypanosomes lack a centromeric histone H3 variant that is essential for kinetochore assembly in all other studied eukaryotes. Trypanosoma brucei possesses large chromosomes that have centromeres and small chromosomes that lack centromeres, yet both types of chromosomes undergo accurate segregation during mitosis. Very little is known about the molecular machinery that segregates these chromosomes. I propose to identify associated factors by PICh (Proteomics of Isolated Chromatin segments) and yeast one-hybrid assays. Identified proteins will be systematically characterized by GFP tagging and RNAi depletion analyses to test whether they have roles in chromosome segregation. I will also identify domains that confer mitotic stability of small chromosomes that lack centromeres. These efforts should lead to an understanding of the segregation machinery in this evolutionary distant eukaryote.

Immune cells play an essential role in protecting the body from invading pathogens by directing pathogen specific responses. These pathogen-tailored responses are largely executed by changes in expression of specific gene programs. Recent studies suggest that mutations in cis-regulatory elements in the toll-like receptors (TLR) pathway affect diverse set of disease. There is currently great need to develop a mechanistic and predictive model of how these dynamic gene responses are generated and controlled.
My proposal addresses this fundamental question with the aim of providing a systematic and dynamic view of the regulatory network underlying the transcriptional response to pathogens. Using a comprehensive, high throughput chromatin immunoprecipitation approach followed by sequencing (HT-ChIP-seq), I will map the genomic localization of all major transcriptional regulators. This physical binding data will be combined with differential gene expression responses under pathogen stimuli in WT cells and following systematic perturbations. The integrated data will allow for quantitative predictive models of gene regulation. I will validate the model by careful genetic perturbation and biochemical purifications of specific factors and complexes predicted by the model.
The successful completion of this project will provide a physical regulatory network under which to understand the impact of misregulated proteins, and a model to analyze the impact of regulatory mutations in the human population. With the help of the CDA award, I hope to have the funds as well as the scientific support of the HFSP community that will enable me to tackle this ambitious goal.

Dendritic spines are small dendritic protrusions (~0.5 µm in diameter) where most excitatory synapses reside. Spine enlargement is associated with, and essential for, induction of long-term synaptic potentiation (LTP), a cellular model for learning and memory. Following LTP induction, the spine volume increases rapidly by 3-4 times within 1 min and decays over several minutes, followed by a smaller sustained enlargement lasting more than 1 hour. This spine structural plasticity requires re-organization of the actin cytoskeleton, and exocytosis and endocytosis of vesicles. However, the detailed dynamics of membrane trafficking and cytoskeleton rearrangements during spine growth are unknown. This project proposes to develop a technique to image structural dynamics of spines with nanometer resolution using high-speed atomic force microscopy (AFM). By resolving the detailed structural changes including filopodia extension, membrane trafficking, and rearrangement of surface proteins during the spine growth, this project is expected to lead to a better understanding of the mechanisms underlying structural plasticity of the spine and LTP, and ultimately learning and memory.

This project seeks to understand how cells migrate in complex environments. While cell migration on flat substrates has been thoroughly studied, cells migrating in vivo are faced with substrates of varying geometry, molecular composition and mechanical properties. Here, the cells show a much richer phenotype and are known to adapt their internal machinery to the extracellular environment. Different cell types exhibit specific migration strategies such that some of them use predominantly actin polymerization-powered protrusions, while others use motility based on pressure-driven blebbing as a default. Furthermore, cells have been observed to switch between these two different migration strategies upon manipulation of the actin polymerization dynamics within the cell, but also upon modification of such substrate properties as rigidity and adhesiveness. We will employ zebrafish germ cells and mouse leukocytes as in vivo model systems that represent the “professionally” blebbing vs. the actin polymerization driven phenotype. In vitro, these and other model cell types will be subjected to genetic, pharmacological and most importantly environmental manipulations that will uncover their range of plasticity regarding force generation and transduction. We will study how the cellular protrusive and contractile activities respond to variations of the geometry, adhesiveness and stiffness of the external substrate and to the presence of external loads. Readouts will include the dynamic visualization of cytoskeletal components, morphological response of the cell and actual motility, while forces exerted by the cell will be recorded quantitatively. Our data will be analyzed in the light of physical models to understand the mechanisms by which internal and external factors affect the strategy of cell motility, and to identify the key physical parameters governing the efficiency of actin-powered protrusions and bleb-based motility. The proposed research will establish novel techniques to quantitatively measure forces and visualize cytokeletal dynamics in a wide range of engineered as well as physiological settings. The integration of experimental data and theoretical modeling should ultimately lead to the development of a universal phase diagram that allows to predict the migratory strategy of a cell as a function of internal and environmental conditions.

Certain types of memories, especially fear memories, are stored in collections of cells in a part of the brain called the amygdala, in a manner that depends on the activity of the transcription factor CREB.

Experiments in which CREB activity was altered in subsets of neurons in the amygdala revealed that enhancement of CREB activity can enhance memory formation in those neurons and inhibition of CREB can decrease it. However, these experiments controlled CREB levels on time scales of days to weeks, whereas a mouse can form memories as a result of a single event lasting a few seconds. Is CREB required precisely during this training event? Soon after? Just before?

Memories, once formed, are not static but may be reconsolidated or undergo extinction depending on the nature of further experience. These processes also depend on CREB, but again it is unclear when CREB is required.

Using a combination of computational protein design, genetic selection via phage display, and structural biochemistry we will create light-switchable versions of CREB (photo-CREB) and a dominant negative form of CREB (photo-DN-CREB). Photo-CREB will allow activation of CREB with blue light irradiation. Photo-DN-CREB will allow inhibition of CREB with blue light irradiation. Both processes will be reversible (i.e., activation or inhibition will cease after the light source is removed). These proteins will be chimeras of the basic leucine zipper (bZIP) DNA binding region of CREB and the light sensitive protein PYP (photoactive yellow protein). These designed and evolved proteins will be analyzed and optimized in vitro using a range of biophysical techniques (X-ray, NMR, DNA binding assays). Experiments with cells in culture will assay kinetics of photoswitching and control of transcription using CREB reporter assays. Light-switchable CREB proteins will then be expressed in freely moving mice with optical stimulation delivered with an optical fiber directly to the mouse’s brain.

This approach using light-switchable proteins will permit the processes of memory formation to be probed on a time scale of seconds to minutes - an unprecedented degree of temporal resolution – and will therefore enable fundamental questions about the role of CREB in memory formation to be addressed for the first time.

The aim of the proposal is to determine how large scale search and taxis patterns of movement are produced by C. elegans at the motor output level and to quantify and predict the adaptive properties of the motor output in the presence of noisy sensory information in spatiotemporally structured environments. Understanding how movement patterns on the ecological scale are produced in response to (or absence of) sensory information is a fundamental, multi-scale, interdisciplinary problem of behavioral and evolutionary biology and biophysics. While most such work has been done by attempting to reduce behaviors to their molecular mechanisms, we propose to take a holistic, integrative approach. Our interdisciplinary team will comprehensively study and model sensory signalling and motor control underlying the adaptive movement strategies of a roundworm C. elegans, with particular emphasis on the thermal information processing system. We will bridge molecular, organismal, and ecological scales by investigating optimality of the behavioral patterns in experimentally controlled environmental contexts, and we will connect neuroscience, behavior, and sensory learning in these animals by characterizing the adaptive responses to the changes in the environment.

C. elegans is a "simple" organism with only 302 neurons, but with complex sensorimotor behavior, which includes nontrivial motion patterns, the ability to find food and to learn by conditionally associating temperature and food. These behaviors are adaptive and are influenced by the statistics of sensory cues in the environment. Our work will focus on answering questions such as: How do physical and statistical properties of the environment affect the worm behavior? Is the worm optimal in utilizing the available information? Can the worm's search patterns be predicted using optimization principles with appropriate physical constraints?

C. elegans is an ideal model for this research. Indeed, to date complete characterization of sensory motor behavior has been achieved, arguably, only for bacteria. However, physics on the microscale severely limits these behaviors, and actions performed by microbes are rather simple. On the other hand, C. elegans displays a wide range of behaviors, including associative learning, a hallmark of higher animals. Yet it is still simple enough to avail a large set of powerful genetic, molecular biology, and optical neurophysiology tools; the entire motor output can be efficiently represented within low dimensional computational model; and the worm environment can be precisely manipulated experimentally. In view of this, C. elegans will become a “hydrogen atom” of the integrated research that connects biophysics of sensory processing to motor behaviors and their adaptations and to ecological consequences of these behaviors. This proposal will be the first in the series developing this atom.

Malaria is one of mankind’s deadliest diseases. It is caused by a parasite that infects the circulating red blood cells, transmitted via the bite of an Anopheline mosquito. To survive and multiply in both man and mosquito, the parasite has evolved many novel biological adaptations to help it pass through the distinct tissues of the two hosts on its infectious journey. Perhaps most striking of all is the speed with which the parasite is able to move through tissues and enter host cells. To do this, it has evolved a molecular engine similar to that in our muscles, made up of two key proteins actin and myosin. However, in parasites these two proteins have diverged radically, making predictions about their function difficult. Actin, a protein that spontaneously comes together to form filaments, is crucial to many cell functions across life. Indeed, because of this property, cells, be they parasite, brain or cancer invest large resources in regulators to control the whereabouts and timing of filament formation. Once formed, these then provide a track for the myosins, the motor proteins of the cell, to run along, pulling cargo or even the entire cell as they go. In malaria parasites the actin-myosin engine is one of the secrets to this unique cells success. We want to understand how the engine works. How the filaments form and where these dynamic events occur inside the parasite cell. To this end, our three teams came together, comprising biologists, chemists and biophysicists, each with different interests in cell motility and parasite biology but united in using advanced technologies to see molecules in action either within parasites or in the Petri dish. However, in combining our efforts we aim to go one stage further. We want to generate fluorescent parasite lines that show us molecules in action in real time as well as mutants that show us how they move when actin regulation goes awry. We also want to build the entire parasite engine in vitro from its defined parts. This not only gives us a platform to test how the engine functions, but also allows us to test drugs for their capacity to inhibit engine parts and potentially halt parasites dead in their tracks. As it reveals key insights into how these deadly parasites move, this program will lay a foundation for new ways to treat malaria disease and rid mankind of one of its greatest scourges.

Understanding the specificity, affinity and dynamics of odor ligand-olfactory receptor interactions represents an outstanding problem of sensory neuroscience, structural and molecular biology. We recently identified a novel family of Drosophila olfactory receptors, the Ionotropic Receptors (IRs), which are homologous to ionotropic glutamate receptors (iGluRs), a conserved family of ligand-gated ion channels with a well-characterized “Venus flytrap” ligand-binding domain (LBD). The primary sequences of IR-LBDs are highly divergent and lack one or more key glutamate-binding residues, consistent with their implication in recognizing diverse small molecules. We propose the IRs as an excellent new model to define the structural basis of odor ligand-olfactory receptor interactions through a combination of Nuclear Magnetic Resonance, X-ray crystallography, molecular genetics and electrophysiology. Our results will provide insights into how a single protein fold can selectively accommodate myriad odor ligands with different affinities and how olfactory receptor repertoires evolve functional diversity. Moreover, the results will open the possibility to engineer IRs of novel ligand-binding specificity, with applications as genetically-encoded chemical sensors.

The discovery that four transcription factors – Oct4, Sox2, c-Myc and Klf4 – can convert somatic into pluripotent cells (iPS cells) has fueled the hope that individualized iPS cell-based therapies will be effective against prevalent diseases such as neurodegenerative disorders, diabetes and cancer. However, a quantitative foundation to the problem of induced pluripotency or reprogramming is missing. We will apply time-lapse microscopy and a powerful single-cell and single-molecule mRNA counting method developed in Dr. van Oudenaarden´s laboratory to state-of-the-art iPS models developed by Dr. Jaenisch´s group at M.I.T. With these tools in hand, we will measure the co-variation of multiple transcripts in thousands of cells exposed to various amounts and combinations of Oct4/Sox2/c-Myc/Klf4 and reconstruct the dynamics of pluripotency transcriptional networks as a function of the cell-cycle with single-cell resolution. In addition, we will count the same transcripts in iPS clones with various degrees of pluripotency to find which transcriptional network configuration associates with the highest potency. The proposed study will advance our knowledge on the molecular mechanisms of reprogramming and set a strong quantitative foundation for engineering large-scale iPS cells production for regenerative medicine.

CD8+ cytotoxic T cells are crucial to combat infectious pathogens. CD8+ T cells recognize peptides presented by MHC class I molecules on the surface of infected cells. The processing pathway for cytosolic proteins is relatively well understood but how exogenous material derived from vacuolar parasites is processed, remains unclear. This question is key for the understanding of host-pathogen interactions
Here I propose to study the molecular and cellular mechanisms leading to presentation of pathogen-derived material by MHC I. We will use the genetically tractable, obligate intravacuolar parasite Toxoplasma gondii (Toxo) as our main model. Toxo is an opportunistic pathogen which infects a large range of hosts including mice and humans.
During my HFSP fellowship, we have identified natural and protective antigens recognized by T cells in Toxo-infected mice. These tools are indispensable for the achievement of this project. We will examine the molecular steps leading to generation of the final epitope from the full-length parasite antigen (aim 1), study how parasite protein trafficking controls access to the MHC I pathway (aim 2) and identify host effectors that regulate organelle trafficking and may be involved in presentation of Toxo antigens (aim 3).
We will use a combination of gene targeting approaches in the parasite and host cell, advanced biochemical methods (mass spectrometry) and novel cellular assays (shRNA library). Our data should reveal new pathways facilitating or interfering with MHC I antigen processing and may ultimately suggest ways to facilitate antigenic generation and enhance parasite immunity.

As mechanical systems, cells control their shape via tight coordination of the dynamic self-assembly of cytoskeletal polymers (e.g. actin filaments) and modulation of intracellular tension exerted among these filaments by molecular motor proteins (e.g. myosins). Highly dynamic and contractile actomyosin-based structures, such as stress fibers of non-muscle cells and sarcomeres of muscle tissue, lie at the heart of fundamental cellular processes including morphogenesis, establishment of polarity and overall motility. Although it is well established that stress fibers play central roles in cell biology, the chemical and physical principles driving the formation of these highly organized contractile structures are largely unknown. The identification of the biochemical mechanisms and biophysical laws that govern the macroscopic organization of the cytoskeleton in cells is rendered difficult by the complexity of the systems. Simplified experimental systems in which actin dynamics can be reconstituted in the presence of regulatory factors and motor proteins are needed to understand the mechanism of stress fiber formation and the consequences of myosin-dependent force generation on the organization and stability of complex actin structures. We will use a multi-disciplinary approach to reconstitute the assembly, turnover, and contractility of stress fibers in vitro and determine how the mechanical and contractile properties of intermediate and assembled structures depend on the individual protein components. Our approach will integrate a new versatile micropatterning method capable of imposing spatial boundary condition to the growth of actin filaments, the use of diverse molecular motors with unique biochemical properties, and the use of AFM and laser trap nanometry to obtain reliable determination of the generated contractile fiber structures and mechanical properties. Theoretical biophysical modeling and computer simulation will complement these experimental works. General principles regarding cell mechanics and the hierarchy of molecular components will emerge from this multi-disciplinary work.

The epidermal growth factor (EGF) receptor family has been widely studied for its role in cancer. Recently, Her3 has attracted much attention because of its pivotal role as preferred dimerization partner for Her2 in Her2-positive breast cancer. It is thus of key importance to not only target Her2, but also Her3. To this purpose, structural and functional data of Her3 and in particular of the Her2/Her3 heterodimer are strongly needed. However, little is known about this heterodimer. Certainly no 3D structure is available: the heterodimer has so far shown to be too unstable to crystallize. Additionally, oligomerization studies would improve the understanding of receptor activation in situ.

X-ray crystallography and single molecule spectroscopy are methods of choice to answer these questions. Novel, alternative approaches will be applied, making use of the enormous potential of designed ankyrin repeat proteins (DARPins) targeting Her2 and Her3. For crystallization trials, the Her2 and Her3 ecotodomains will be produced with a C-terminal IgG-hinge to induce heterodimerization via disulfide bonds. DARPins can facilitate crystallization by acting as chaperones. Receptor oligomerization can be studied by applying Alexa-labelled DARPins in two colour coincidence detection microscopy (TCCD). Coincident fluorescence from single molecules labeled with two different fluorophores will be detected, as the molecules diffuse through a confocal volume. Finally, the potential biological effect of Her3-selective DARPins on tumor cells will be examined.

These data will extend our knowledge of tumorigenesis and aid in the development of new, much needed cancer therapeutics.

Many Nobel Prize winning advances in biology were driven by x-ray studies of crystallized biomolecular systems. A recent revolution in x-ray sources, the x-ray free electron laser (XFEL), eliminates the radiation damage problem that plagues studies using x-rays and electrons. XFELs open a new horizon of atomic resolution study of non-periodic biological structures. The Linac Coherent Light Source, LCLS, at the SLAC National Accelerator Laboratory recently became the first operational hard x-ray FEL. Its x-rays are a billion times more brilliant than any other source. One of the most promising scientific applications of XFELs is coherent diffraction imaging (CDI) of non-crystalline biological samples at sub-nanometer resolution. The basis of this "diffract-and-destroy" method is the recent observation that a sufficiently brief x-ray pulse provides a useful diffraction pattern before significant radiation damage commences. Our international team is comprised of the requisite expertise to uncover new scientific discoveries in complex non-periodic biological systems using XFEL experiments. We have i) extensive experience in algorithms for CDI, ii) over six years of experience in XFEL experiments, including LCLS, iii) the requisite biological expertise for virus preparation and data interpretation, and iv) unique experimental apparatus. Our primary objective is to achieve the highest resolution structure of algal (PBCV-1) viruses. Little is known structurally about the algal virus PBCV-1. 3D reconstructions of PBCV-1 at 2.2 nm resolution exist but many questions remain open. These include, for example, how the genome is packed and the structure of the unique vertex involved in infection. Since single particle CDI is so new, significant effort will be directed toward method development. At present, there are at least two computational methods in the literature that we will use for determining the relative orientations of diffraction patterns, permitting a 3D data set of diffraction intensity to be assembled for phasing and image reconstruction. XFEL-CDI methodologies are now on the verge of contributing to the biological questions posed for many viruses, including PBCV-1. We were awarded LCLS beamtime to begin addressing these questions and will use HFSP support to help stimulate new biological frontiers by the x-ray imaging of viruses.

Many proteins and enzymes frequently undergo structural rearrangement to execute their functions. Ligand/substrate entry and binding are often associated with some degree of localized disorder of both protein and solvent. Characterization of these states is vital to understand the mechanisms of general biological functions. In this agenda I will use human zinc dependent metalloproteinases, key physiological enzymes, as model system to study the correlation between protein and solvent motions with enzyme function. Here, I address the question of the enzyme-solvent-substrate interplay with special focus on the active site structural kinetics during the hydrolysis of peptide substrates and on the role of the omnipresent solvent: water. Specifically, I will combine stopped-flow x-ray and THz structural spectroscopic analyses to quantify both active site structural-kinetic transitions with coupled solvent-protein motions in real time and at atomic resolution. These studies will be complemented with solution NMR and computational analyses. Applying this multidisciplinary approach will allow exploring general and yet unknown aspects in enzymatic catalysis such as the effect of catalytic metal ions on solvent water dynamics, the correlation between localized active site chemical transitions with solvent dynamics and the influence of enzyme side chains in prearranging approaching substrate molecules to a preferential conformation for binding together with solvent molecules. Thus establishing a novel experimental procedure as well as revealing the detailed mechanism of metalloenzyme molecular recognition, onset of turnover and product release will be the ultimate goal of my agenda.

The primary virulence strategy utilized by many bacterial pathogens is the type III secretion system (T3SS), a molecular syringe through which effector proteins are injected into host cells. These effectors modify the host cell to promote bacterial infection, and as a result are critical determinants of pathogenesis. The study of effectors and their host targets is providing valuable insights into mechanisms of both pathogenesis and host immunity.

Despite the intensive investigation of effector targeting mechanisms, host targets for the majority of effectors remain unknown. This is largely due to the reliance upon target identification strategies, such as the yeast two hybrid assay and coimmunoprecipitation, which require isolation or over/ectopic-expression of the effectors and their proposed targets. The artificial nature of these approaches can prevent the observation of effector-target interactions.

Due to these limitations, I propose a novel strategy that employs a pathogen encoding effectors with photocrosslinking (PCL) unnatural amino acids for the in vivo detection of host targets. In this approach the genetically modified pathogen injects PCL-effectors into the host via natural infection. These PCL-effectors interact with cognate targets in their physiological environment. Ultraviolet light irradiation of the infected cells stimulates PCL in vivo, yielding covalently coupled effector-target complexes that are recovered and analyzed using affinity purification and mass spectroscopy. This in vivo tool will allow for the identification of the most relevant host targets and thus promises to advance our understanding of pathogen-host interactions.

The malaria parasite has developed an unusual mechanism to regulate gene expression, which seems largely dependent on epigenetic modifications such as chromatin remodeling. One of the main mechanisms involved in chromatin remodeling is DNA methylation. Although previous studies have been unable to detect DNA methylation in P. falciparum, recent data from the Le Roch lab obtained using the latest and most sensitive next-generation sequencing technology show that the genome of P. falciparum is in fact highly methylated. In the current project, I will investigate the functional role of DNA methylation in controlling malaria parasite development. First, I will determine DNA methylation patterns throughout the asexual blood stage and in sexually mature gametocytes, and relate these to changes in nucleosome positioning and mRNA transcript levels. Second, I will explore whether DNA methylation is involved in transcriptional regulation of the var genes that are responsible for parasite survival and immune escape. Third, I will characterize the cellular localization and biochemical activity of a putative DNA methyltransferase and determine whether this enzyme is essential for the development of the malaria parasite. In addition, I will perform pull-down experiments to identify proteins interacting with this putative MTase. Together, these studies will contribute toward a more comprehensive understanding of the complex process of transcriptional regulation in P. falciparum.