George N. Phillips, Jr., Ph.D. - US grants

Muscle contraction is switched "on" and "off" in response to external chemical or electrical signals. Within each muscle cell activation is controlled by calcium ions and regulatory proteins: at high levels of calcium the muscle is "on", at lower levels the muscle is "off". This calcium switch consists of the proteins tropomyosin and troponin. Using X-ray crystallography together with electron microscopy I propose to determine the detailed three-dimensional structure of tropomyosin, visualize its interactions with troponin in the crystal lattice, and prepare crystals of troponin and/or its subfragments for high resolution structure determination. This information will allow us to better understand how these proteins control contraction. Motions of tropomyosin have been postulated to play a critical role in the regulatory process. The flexibility of tropomyosin in the crystals will be examined by analyzing the diffuse X-ray scattering in conjunction with crystallographic studies at various temperatures. These results will allow us to determine the kinds of motions and conformations which tropomyosin can adopt during the regulatory process. Although tropomyosin was first discovered in muscle cells, we have recently recognized that this protein plays an important role in the cytoskeleton of many cells. Moreover, tropomyosin-like proteins have been found at the surface of certain pathogenic bacteria, and appear to be critical factors in determining their virulence. Attempts will be made to characterize the molecular structure of one of these proteins from streptococcal bacteria. Knowledge of how this "M protein" works could be significant in developing ways to control streptococcal diseases.

The goal of this project is to determine three-dimensional structures of several muscle proteins, with special emphasis on those involved in the regulation of muscle contraction, and to relate the resulting structures to physiological events. Tropomyosin and troponin are key components of thin-filament based regulation in skeletal muscle and tropomyosin is also found as a component of actin filaments in a range of smooth muscle and non-muscle cells. We are proposing to complete the structural analysis of tropomyosin to the limit of resolution of the crystals, which is about 3.5 angstroms at present. We will construct an atomic model for the protein, and refine it using restraining methods and/or molecular dynamics. The resulting structure will be related to its role in muscle regulation. We will also determine low resolution structures of complexes of tropomyosin and troponin and fragments of actin in the Bailey crystals at low and high calcium concentrations, to reveal gross changes in the regulatory complex during activation. We are proposing to solve the structure of smooth muscle regulatory complexes, including the tropomyosin-caldesmon complex at low (17 angstrom) resolution to reveal the attachment site of caldesmon on tropomyosin. We will also bind caldesmon fragments containing the putative tropomyosin binding regions of caldesmon in crystals and determine their binding sites on tropomyosin. The results will be compared to those of troponin-containing tropomyosin crystals. We will also determine the locations of the AMP and ATP sites in adenylate kinase, and resulting conformational changes that result on substrate binding, by completing the structure of this enzyme to 2.2 angstroms resolution. The significance of this work is two-fold. The enzyme is a key player in the "energy charge" of the cell, catalyzing the reaction MgATP + AMP <--> MgADP + ADP. Furthermore, adenylate kinase is a close "relative" of one domain of the cystic fibrosis gene product, and although not implicated in cystic fibrosis, the study of adenylate kinase may reveal aspects of the function of the cft protein.

Dr. Phillips will examine the dynamics of myoglobin by a newly developed experimental method. Analysis of the diffuse X-ray scattering will reveal the range and distribution of conformational states of the molecule. The methods developed in this project for analyzing the diffuse X-ray scatter from myoglobin crystals will also have broad application to other macromolecules. Protein structure and dynamics are intimately related. X-ray crystallography has been a powerful tool for determining the average conformation of macromolecules. Techniques such as NMR and fluorecence spectroscopy have provided detailed knowledge of the dynamics of targeted "parts" of protein molecules, but not a good overall description of the motions of the entire protein. Theoretical calculations can predict dynamic behavior of proteins, but as yet are limited to simulations in very short periods of time. Their connection to reality is still somewhat tenuous. This project will help bridge the gap between theoretical studies of protein dynamics and experimental determinations of the real behavior of the protein.

Myoglobin and hemoglobin have been and will continue to paradigms for developing our general understanding of protein structure and function. The work propose here will provide crystallographic data on the interaction of ligands of different sizes and shapes with myoglobin and hemoglobin, and examine the structural effects of changes in key amino acid side chains involved in ligand binding. key determinates of the affinity of proteins for their ligands include covalent and hydrogen bonds, electrostatic forces, nonbonded steric and van der Waals interactions, solvent, entropic affects. To date, non complete accounting of all of these "forces" has been made for ligand binding to any protein. Because of its small size, relative simplicity, and extensive experimental study, myoglobin is an ideal protein for this endeavor. Protein with the amino acid sequence of sperm whale myoglobin has been produced in E. coli by Dr. Sligar and co-workers. The only significant difference is that the initiator methionine remains after translation. This expression system allows the rapid generation of mutant proteins to be studied in the proposed research. The structure of the "native" protein has recently been determined so that the successful bacterial synthesis of myoglobin has been verified. Two amino acid side chains are critical for ligand entry and binding in myoglobin. These amino acids are in the distal pocket at positions 64 (HisE7) and 68 (ValE11) in the sequence. The proposed high resolution X- ray crystallographic structure determinations of the deoxy forms of the HisE7->Gly and HisE7->Phe mutants will provide key data on the steric factors that affect ligand entry to the binding pocket of myoglobin. The role of these side chains in the determination of ligand specificity will also b studied by determination of the structures of carbonmonoxy and oxy forms of HisE7->Gly, HisE7->Gln, HisE7->Leu and HisE->Phe mutants, and the carbonmonoxy structures of ValE11->Ala, ValE11->Ile and ValE11->Phe. The effect of different sizes and shapes of ligands on the native forms of myoglobin and hemoglobin will also be examined by determining the structures of several alkyl isocyanide complexes of these proteins. The resulting "strain" on the proteins will indicate which parts of the binding pocket are involved in discrimination between different ligands. The end result of the proposed research will be a better understanding of the details of ligand binding in heme proteins. Molecular dynamics calculations are designed to simulate the detailed dynamic behavior of proteins, and to quantitatively predict some of the forces enumerated above. For these calculations to be meaningful, however, they must be carefully checked against experimental data whenever possible. As the structures of the various myoglobin-ligand complexes are determined, we will use molecular dynamics programs to attempt a rationalization of the changes in structure with the changes in kinetic and thermodynamic measurements.

This award to a group of 27 Faculty will provide positions for 5 graduate students in a new computational biology training program at the Center for Computational Biology established jointly by three institutions (Rice University, Baylor College of Medicine and the University of Houston). The faculty come from Departments of Cell Biology, Biochemistry, Chemistry, Computer Science, Statistics and Electrical Engineering. A few also participate in the STC for Research on Parallel Computation at Rice University. The training program will emphasize visualization, algorithm development and advanced computation in biology, biochemistry and biophysics. Also included are courses offered by the individual institutions, a course taught jointly by faculty from all three, and a Center-sponsored seminar series. The award includes funds to be used to aid in recruiting and to promote student participation in meetings & workshops.

9413229 Phillips This award to large group of outstanding faculty from Rice University, Baylor College of Medicine and the University of Houston provides funds for support of a training program on computational aspects of modern biology, including experimental and theoretical analyses of macromolecular structure using crystallographic, microscopic and other experimental techniques, and computational analysis of genomic structure. The faculty groups from each institution provide complementary skills in biochemistry, biophysics and computer science, resulting in a program broader and stronger than could be provided by the individual institutions. Important aspects of the training include dual mentorship for postdoctoral trainees, a large summer undergraduate program and a biannual national symposium on selected topics in computational biology. This award is also supported by the New Technology Program of the Division of Advanced Scientific Computing and by the Computational Biology Activity of the Division of Biological Instrumentation and Resources. ***

9315840 Phillips Dr. Phillips proposes to develop a strategy for the complete refinement of macromolecular structure and dynamics in crystals by utilizing all of the available X-ray scattering data, namely, the low and high resolution Bragg reflections, but more importantly the diffuse scattering between Bragg peaks. In addition to the average structure then, the result of this expanded refinement will be a picture of the conformational states available to a macromolecule, knowledge indispensable for correlating molecular structure and dynamics with biochemical function. Although generally applicable to any macromolecular crystal, the methods will initially be applied to the oxygen binding protein myoglobin, the nucleic acid transfer RNA, as well as the enzyme aspartate aminotransferase where disorder in the molecule's ligand binding domain can be controlled in crystallum. X-ray diffraction data will be collected on imaging plates, which afford the dynamic range necessary for collecting the weak diffuse scattering signal, and for placing this signal on the same absolute scale as the much more intense Bragg data. Molecular dynamics calculations will play in integral role in the extended refinement of the Bragg and diffuse intensities; thus high performance computing technologies will prove a useful tool. %%% The microscope has always played a role in science, particularly biology. The more detail visible, the deeper our understanding of the mechanics of life. Even the most powerful glass lenses cannot image atoms however. To see what life looks like down at the level of atoms and molecules, X-ray crystallography is used. In this technique, high intensity X-rays are scattered off a sample; then sophisticated computer algorithms are employed to analyze the results, acting as 'mathematical lenses' to form pictures of the arrangement of atoms in the biological structure. X-ray crystallography has had an enormous impact on our understanding of the atomic stru cture of the biological world, from the earliest structure of the genetic code DNA to the structure of enzymes involved in the replication of the AIDS virus. Nevertheless, until now X-ray crystallographic researchers have only analyzed part of the signal from their scattering experiments, namely, the data corresponding to the average, static picture of a biological molecule. Our atomic views of life are consequently motionless at present. Watching a butterfly or a ballerina, however, we all know that what makes living organisms special are their movements. Thus, we propose developing methods to analyze all of the X-ray data, the traditional part due to the average atomic structure as well as the part due to atomic movements. This will lead to dynamic pictures of living processes down at the atomic level. The proposed study, though primarily motivated by questions in biology, will require new understanding in the physics of X-ray scattering as well as substantial high performance computing technologies. The result promise an increased understanding of life at its most fundamental level. ***

; R o o t E n t r y F ZpÕ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l ZpÕ ZpÕ 0 ! " # $ % & ' ( ) * + , - . / 1 2 3 4 5 6 7 8 9 : ; < = > ? @ A B C D E F G H I J K L M N O P Q F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; e = e z z p p p p p p z & 1 x W T 3 & p & p p p p p x p p p p . Estimation of genetic relatedness, including identification of parents, is central to many kinds of biological studies. Relatedness and parentage can both be estimated through the use of genetic markers, though the rarity of good markers has limited this approach for many organisms. However, new kinds of markers, including multi-locus fingerprints, minisatellites, microsatellites, and RAPD's, now make accurate kinship assessment a practical goal for just about any species. This award will result in two computer program becoming generally available to the research community. The first, RELATEDNESS, focusing on statistical estimates for genetic relatedness, will be a rewritten and restructure version of a program that has been available for several years. The second program, PARENTAGE, will be specifically for paternity and maternity analysis. It will be designed to make maximum use of the different kings of prior information available in studies of vertebrates, social insects, plants and other organisms. This award is being supported by the Computational Biology Activity, Animal Behavior, and Population Biology programs. { S u m m a r y I n f o r m a t i o n ( Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT Estimation of genetic relatedness, including identification of parents, is central to many kinds of biological studies. Relatedness and parentage can both be estimated through the use of genetic markers, though the parity of good markers has limited this Bertha L. Luster Bertha L. Luster @ G Õ @ @ G Õ @ Microsoft Word 6.0 2 ; y z u c y z !. ! K @ Normal a c " A@ " Default Paragraph Font { z . Bertha L. Luster \\CLM2\BIRWORK\QUELLER.ASC @HP DeskJet 500 LPT1: HPDSKJET HP DeskJet 500 D L f , , d s HP DeskJet 500 D L f , , d s 1 Times New Roman Symbol & Arial " V h { e { e $ 3 Estimation of genetic relatedness, including identification of parents, is central to many kinds of biological studies. Relatedness and parentage can both be estimated through the use of genetic markers, though the rarity of good markers has limited this Bertha L. Luster Bertha L. Luster ; ;

The general goals of this project are to establish solid structure- function relationships in ligand binding to myoglobin that include definitions of the roles of key amino acid residues, the roles of dynamics and conformational substates, and the role of water. Because of the accessibility of myoglobin to many spectroscopic techniques and the plethora of studies on this molecule, a unique opportunity exists to develop a complete biophysical understanding of the detailed ligand binding pathways. Using X-ray crystallography to determine three- dimensional structures and using computational tools such as electrostatic potential calculations and molecular dynamics simulations based on these structures, correlates between structure and function will be developed in a way not possible with other systems. In particular, the aims are to: 1) Determine structures of mutants designed to test ideas about the physiological function of myoglobin. 2) dynamic details of ligand binding pathways in mutants using cryo- crystallography. 3) Develop conformational substate models of key myoglobin complexes 4) Use electrostatic potential calculations to probe the role of charge and polarity on ligand orientation and binding 5) Use molecular dynamics simulations to calculate and then predict binding energies of ligands 6) Determine the crystal structures of chemically modified myoglobins to test specific theories on the role of metal movements in ligand binding and to answer other questions about the plasticity of the distal pocket. 7) Resolve questions about the details of CO binding in crystals versus sol ion 8) Determine the structures of mutants designed to test ideas about the folding and stability of the protein. The insight gained in the study of myoglobin also has a direct medical application. In the development of hemoglobin for a cell-free blood substitute, there are two factors where the results can and will be applied. First, the oxygen affinity must be re-tuned for a cell-free hemoglobin, and this can be achieved with mutations at the distal histidine position. Secondly, the protein must keep its heme tightly bound and reduced within the globin to be functional, and these studies on heme loss, mechanisms of oxidation and globin stability will help in the design process. Thus, the last specific aim is to: 9) Determine the structures of mutant hemoglobins and determine the extent to which myoglobin can serve as a simple model system for the development of blood substitute proteins. This grant application represents the structural components of a collaboration with Prof. John Olson and members of his laboratory, where the mutants are constructed, and their kinetics and other spectroscopic properties are measured. The two groups work jointly to design the mutants, to train students and postdoctoral trainees in all techniques used in both laboratories, and to synthesize meaningful structure-function correlates.

We believe that a comparison of proteins that are very closely related in sequence, but have different temperature/activity profiles will most likely yield useful information on the origins of ADK thermostability. On this basis we have proposed to carefully compare the three dimensional structures of three adenylate kinases . Station 14 BM-C was used to collect data under cryogenic conditions at a single wavelength close to I A.

`The PIs will investigate and develop software and computational methods to address one of the most challenging current problems in molecular dynamics (MD) simulations - large proteins, consisting of tens of thousands of atoms in solution over the physiological range of at least a microsecond of folding time. This will be accomplished using a reduced basis approach based on singular value decomposition (SVD) of the MD trajectory.

The work will entail:

- developing a block SVD updating scheme that will enable new trajectory information to be adjoined to a current truncated SVD approximation to a prior trajectory and avoid having to store the entire trajectory
- developing, analyzing, and implementing a reduced basis integrator that will work in concert with the SVD updating scheme to compute the reduced basis simulation more rapidly
- adapting the fast marching algorithms developed for latent semantic indexing to develop the rapid graphical query tools to locate sites that potentially match local structures of interest
- developing the I/O support and visualization capabilities to handle the extremely large data manipulation and representation problems that will be generated

The result of the research will be an efficient time integration scheme that can drastically limit storage and yet resolve detail on multiple scales. It will be demonstrated on a fully solvated protein molecule over a time scale of a microsecond, and the high order, low frequency motions will be visualized.

9986534
Colvin

This proposal seeks to exploit the vast effort in protein crystal growth technology in the formation of mesoporous films. The use of these bio-crystals in x-ray crystallography requires essentially perfectly ordered samples, with long-range periodicity extending up to ~1 millimeter. Protein crystallographers have been successful in growing these samples for more than 1000 different proteins. Once grown, such crystals can be used as templates for a range of different materials, by adapting the templating techniques which have been demonstrated in colloidal crystals or surfactants. This will lead to the formation of nearly ideal ordered mesoporous materials, in a planar format suitable for optical applications. These materials will be excellent candidates for x-ray photonic band gap systems.
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Divisions of Chemistry, Materials Research, Bioengineering and Environmental Systems, and Molecular and Cellular Biosciences support this multidivisional award to William Marsh Rice University. This Nanoscale Interdisciplinary Research Team (NIRT) proposal was submitted in response to the solicitation "Nanoscale Science and Engineering" (NSF 00-119). Under this project, Vicki Colvin, a material chemist with experience in template chemistry, Daniel Mittleman, an optical engineer and George Phillips, a biologist with expertise in protein crystal growth, will develop crystalline and monolithic nanostructured metal and ceramic materials using crystals of streptavidin, lysozyme and related proteins. These protein crystalline structures will be reinforced by glutaraldehyde cross-linking before the templating. X-ray diffraction studies and atomic force microscope imaging will be used to characterize the prepared materials with respect to crystal symmetry, periodicity and overall quality of the crystal arrangement. These porous and three-dimensional open crystalline inorganic structures with nanometer spacing may find applications in diffraction gradients, lenses, mirrors and other devices for soft X-ray optics. The research program will also provide a rich multidisciplinary education and training opportunities in material and protein chemistries to postdoctoral, graduate and undergraduate students.

Under the award, ordered mesoporous nanostructured metal and ceramic materials will be fabricated using cross-linked protein crystals as sacrificial templates. The broader impact of the project is the understanding the formation of nanostructures, the templating effect of different protein crystal scaffolding, and the determination of optical properties of the mesoporous structures prepared in the soft X-ray range of the spectrum. In addition, the research program will provide a rich multidisciplinary education and training opportunities in materials chemistry, protein chemistry and optics to graduate and undergraduate students.

DESCRIPTION (provided by applicant): We propose a bioinformatics training program for 16 pre and 4 postdoctoral students per year focused in the departments of Computer Sciences, Biochemistry, Statistics, and Genetics at the University of Wisconsin. Computer scientists with knowledge of biology will join biologists with strong quantitative backgrounds, in a cross-disciplinary approach, with each student having a major and a minor mentor. We will build on a history of computation/biomedical collaborations, which over the years have trained several leaders of the current generation of bioinformaticists. Students with strong quantitative skills and an interest in biology will be recruited mainly from applicant pools of primary and associated departments. Training will be rigorous but tailored to the students' needs and interests. New courses in bioinformatics and genomics recently started by trainers will be an integral part of the training, but close project-oriented mentoring by trainers will form the core training experience for the student. Predoc support of three years and postdoc support for two years is proposed. The P.D., Professor George Phillips, recently served as Scientific & Training Director of the NLM-supported Keck Center for Computational Biology at Rice University, and brings a wealth of experience with the type of training program envisaged. The Co-P.D.'s, Jude Shavlik, Professor of Computer Sciences, and Fred Blattner, Professor of Genetics, have for many years participated in cross disciplinary training in bioinformatics on campus and, through their strong network of interconnected associations at U.W. Madison, will help to firmly anchor and balance the program in its biological and computational aspects. Administration will be through the Genome Center/Biotechnology Center with the able financial/administrative skills of Deborah Faupel and the educational/administrative skills of Dr. Louise Pape, who is a sensitive student adviser, researcher and teacher. Computer assets of the Sun Center of Excellence in Genome Computing, the Condor "grid computing" resource including hundreds of workstations, the advanced chip facility, and the DNA sequencing facility located in the Genome Center will provide core resources. A new building housing the Genome Center is being constructed.

functional /structural genomics; Arabidopsis; plant proteins; biomedical resource;

DESCRIPTION (provided by applicant): The Computation and Informatics in Biology and Medicine (CIBM) training program is helping to produce the next generation of researchers of biomedical problems with strengths in both informatics and biology. The interplay between computational and statistical methods in the biomedical sciences continues to expand rapidly so that now both computer modeling and informatics play key roles. Our faculty and trainees have been developing robust algorithms for the analysis of molecular data. Increasingly, questions in the biosciences are being phrased for these more quantitative approaches, and computer scientists are discovering new computational approaches in attempting to address biological problems. Furthermore, the power of new hardware, algorithms, and software is transforming our thinking about complex systems research. These advances are only possible when computer scientists understand enough about the problems to design usable tools and when bioscientists understand what is possible using computational and information technologies. CIBM has focused on the development of novel bioinformatics algorithms to analyze molecular data, including genome sequences, proteins (levels, interactions, structures), and regulatory pathways. We propose that for phase two of the program that we continue this focus and to further distinguish our program by adding a unique translational medicine component. In a collaboration with the Marshfield Clinic, our trainees will have the added opportunity to develop algorithms to predict clinical parameters, such as disease susceptibility or treatment response, from combined molecular and clinical data. A strong training program, including a multidisciplinary core curriculum has been developed at CIBM, and the environment at the University of Wisconsin in biology and computational sciences provides a rich setting for research training at the graduate and postdoctoral level.

DESCRIPTION (provided by applicant): Natural products and their derivatives continue to play an important role in the drug pipeline. Over time, 7000 known structures have led to more than 20 commercial drugs, and about half of the new drugs approved in the last decade are based on natural products. Screening of new natural products and their analogs will continue, and be enhanced by modern methods such as metabolic engineering, synthetic biology, and structural analysis of compounds and the enzymes that produce them. For example, new biosynthetic routes are being built around engineered systems such as the modular polyketide synthases to produce new compounds. There are also still a tremendous number of new microbial natural products to be explored, as evidenced by the genomic sequencing data coming forth. Nevertheless, the discovery of new compounds remains an adventitious endeavor. Genome mining efforts to identify interesting clusters and to predict what natural products might come from these clusters of genes are beginning to produce hypotheses; however the homology of the enzymes to known enzymes is generally low. We propose to create a new partnership, named the Natural Products Partnership, within the PSI Biology Network framework to play strong joint roles in both the identification of new natural product pathways and the subsequent discovery of new natural product-based pharmaceuticals by revealing the structures and active sites of novel enzymes, characterizing the enzymatic reactions of the gene products, identifying new natural products and thus offering opportunities to identify and customize the pathways by altering specificities and/or identifying novel proteins or domains with desired enzymatic properties. The combination of the PSI Network Centers with their high-throughput structure determination capability and the biology-driven team of natural products scientists through the Natural Products Partnership are strongly positioned to create a high impact program. The experience of the structural biologists with PSI activities and their leadership in developing technologies and infrastructure in the PSI program will make a smooth transition to PSI Biology activities possible.

? DESCRIPTION (provided by applicant): Methyltransferase (MT)-catalyzed S-adenosyl-L-methionine (AdoMet)-dependent methylation is essential to all walks of life and alterations in methylation-dependent processes have direct relevance to microbial/fungal/viral pathogenesis and human disease. Yet, for many MTs, there is a lack of correlative fundamental knowledge regarding specific MT function and corresponding impact upon cellular fate and/or pathogenesis/disease. In addition, while simple chemical or MT-catalyzed methylation of a drug/drug lead can dramatically impact its corresponding ADMET (absorption, distribution, metabolism, excretion and/or toxicity), the structural complexity of many natural products often prohibits doing so in the context of natural products-based drugs/lead development. This proposal seeks to develop general chemoenzymatic alkylation strategies and reagents for that are expected to broadly facilitate the fundamental study, annotation and application of MTs. A centerpiece to the proposed universal platform development is the study, engineering and application of permissive methionine adenosyltransferases (MATs) and MTs, where the model MTs selected represent broad catalytic diversity (C-methylation, O-methylation and N-methylation) and directly act upon a selected set of complex natural product-based drugs, validated clinical candidates or marketed agricultural products. The proposed studies will integrate the chemical synthesis and application of unique methionine (Met) analogs, MAT/MT structure determination, high throughput MAT/MT assay development/application, structure-guided MAT/MT directed evolution, microbial strain engineering, complex natural product (NP) structure elucidation and bioactivity assessment for NP analogs generated. The anticipated outcomes of this study include highly permissive/proficient MATs/MTs engineered for medicinal chemistry applications, novel functional AdoMet orthologs designed as alternative alkyl donors and/or with improved stability, an expanded understanding of MAT/MT structure-activity relationships of potential relevance to MAT/MT inhibitor design, single vessel chemoenzymatic strategies to enable complex NP differential alkylation, engineered microbial strains to enable complex NP differential alkylation and functional MT annotation, and unprecedented differentially-alkylated NP analogs with potential therapeutic and/or agricultural applications. Within this context, the proposed studies will also provide the first bioorthogonal strategy to functionally annotate, interrogate or exploit a single MT within a cell containing a full complement of competing native MTs. While NP methylation has been selected as the model for platform development, it is important to note that reagents and concepts developed will likewise enable the similar study, annotation and application of other class I MTs relevant to cellular development, human disease or microbial/fungal/viral pathogenesis.

ABSTRACT The 10-membered enediynes [exemplified by calicheamicin (CLM), esperamicin (ESP) and dynemicins (DYN)] are arguably among the most renowned natural products (NPs) discovered to date by virtue of their unprecedented complex molecular architectures, notable anticancer and anti-infective potencies and, in the case of CLM, demonstrated clinical utility. The current study builds on a longstanding collaborative effort of achievement and discovery relating to key aspects of 10-membered enediyne biosynthesis as well as parallel innovative efforts to co-opt key biosynthetic catalysts for synthetic applications. The studies put forth will take advantage of this strong foundation and a powerful combination of genetic, biochemical, chemical and protein structural tools to elucidate the remaining unusual biosynthetic transformations and to exploit select catalysts for enediyne non-native modification. Specifically, aims 1 and 2 will focus on extending our understanding of the fundamental steps of enediyne core biosynthesis common to CLM/DYN/ESP, DYN anthraquinone biosynthesis and a selected set of unique tailoring reactions (CLM/ESP thiosugar sulfur installation and aminopentose N- alkylation, ESP C6-hydroxylation and O-glycosylation). In parallel, aim 3 will focus on tactical structural studies to augment both aims 1 and 2 and the structural study of ?unknowns? to facilitate functional annotation. Additional studies in aim 2 with key catalysts and corresponding non-native substrates are designed to assess the potential for strategic installation of chemoselective handles to enable novel approaches for facile, mild bioconjugation of CLM to tumor-targeting mAbs (in collaboration with Pfizer).