Plenary Speaker

Tatiana Kutateladze







Title: Molecular mechanisms of the epigenetic regulation.
Tatiana Kutateladze, Ph.D.
Professor, Department of Pharmacology
University of Colorado Anschutz Medical Campus
Aurora, CO

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045

Plant homeodomain (PHD) fingers comprise one of the largest families of epigenetic effectors capable of recognizing PTMs (posttranslational modifications) of histones. Here, I summarize the structures and binding mechanisms of the PHD fingers that select for modified and unmodified histone H3 tails. I will compare the specificities of PHD fingers, Tudor and other histone readers, and discuss the significance of crosstalk between PTMs and the consequence of combinatorial readout for the selective recruitment of these effectors to chromatin.

Invited Speakers

Moriah Beck







Title: Cell motility:  integrating protein structure and flexibility with cytoskeletal dynamics
Moriah Beck, Ph.D.
Assistant Professor, Department of Chemistry
Wichita State University
Wichita, KS

Actin-based cell motility shapes multicellular development and contributes to numerous diseases, including cancer and cardiovascular disease. Palladin is a recently discovered actin binding protein that plays a key role in both normal cell migration and invasive cell motility, yet its precise molecular role in organizing the actin cytoskeleton is unknown.  Palladin functions as both a molecular scaffold for multiple actin-binding proteins and as a direct link to actin through binding via its immunoglobulin-like domain (Ig3). I will discuss our current progress in utilizing NMR spectroscopy, microscopy and kinetic assays to investigate the interaction between palladin and actin.  The affects of mutations, lipid binding, and phosphorylation on palladin structure and function will be presented, where the goal is to understand how palladin regulates actin polymerization.

Xiao Heng







Title: NMR studies of the conserved 3’X tail of the Hepatitis C virus genome RNA.
Xiao Heng, Ph.D.
Assistant Professor, Department of Biochemistry
University of Missouri-Columbia
Columbia, MO

Hepatitis C virus (HCV) is a single-stranded RNA virus that utilizes its positive-sense genomic RNA as templates for both viral protein translation and minus-strand RNA synthesis. The highly conserved 98-nucleotide element on the 3´-terminus of the HCV genomic RNA, termed as 3´X, plays a critical role in regulating these two functions. Several secondary structures have been proposed for 3´X by chemical probing, enzymatic probing, and phylogenetic studies. However, it is difficult to identify a mechanism imbedded in the structures due to the lack of accurate 3´X structural information. We have employed novel NMR techniques, including long-range Adenosine interaction probing and site-specific deuteration to investigate the structure of 3´X. Our data show that 3´X adopts alternate structures, suggesting an RNA switch mechanism in HCV that fine-tunes viral functions.

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Adina Kilpatrick


















Title: Thermodynamic and structural analysis of calmodulin interaction with the skeletal muscle ryanodine receptor
Adina Kilpatrick
Assistant Professor, Department of Physics
Drake University
Des Moines, IA

In skeletal muscle, the calcium sensor calmodulin (CaM) plays a key role in excitation-contraction coupling, the process of translating neuronal stimuli into mechanical contraction of muscle, by regulating the opening and closing of the calcium channel ryanodine receptor (RyR1). By interacting with this channel differently at high and low calcium, CaM acts as a feedback regulator of calcium levels during muscle contraction: at low calcium, CaM weakly activates RyR1, while calcium-CaM inhibits it. We are investigating the interaction between CaM and its established binding site on RyR1 (CaMBD, residues 3614-3640) using a multi-faceted approach combining biophysical (fluorescence spectroscopy) and structural (solution NMR) methodologies. Förster resonance energy transfer (FRET) experiments in an auto-fluorescent biosensor construct (YFP-CaMBD-CFP) enabled us to determine Gibbs free energies of binding in the absence and the presence of calcium. Using this system, we systematically explored the thermodynamics of molecular recognition between the two biomolecules in high and low calcium environments, as well as the roles played by individual RyR1 residues and each CaM lobe at the interface. To gain additional insights into the interplay between the processes of calcium- and target-binding to CaM, we analyzed the interaction between wild-type or mutated RyR1 CaMBD sequences and CaM mutants in which the Ca2+-binding sites in one domain had been rendered non-functional. Overall, these experiments show that CaM C-domain binding to molecular determinants in the N-terminus of RyR1 CAMBD dominates the interaction, both in the presence and the absence of calcium. However, the interaction is three orders of magnitude stronger at high calcium levels. To obtain residue-specific information of the binding interface, we are currently undertaking NMR studies of isotopically labeled CaM (wild-type and Ca2+-binding mutants) in the absence and presence of wild-type and mutated RyR1 sequences. Preliminary data reveal differential effects of CaM and RyR1 mutations on the interaction between the two biomolecules.

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Jed Lampe
















Title: Exploring the ligand binding conformational sub-space of cytochrome P450 enzymes utilizing a combined approach of NMR, fluorescence, and x-ray crystallography
Jed Lampe, Ph.D.
Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics
University of Kansas Medical Center
Kansas City, KS

Cytochrome P450 (CYP) enzymes utilize a flexible protein backbone to access large regions of conformational sub-space to conduct a variety of oxidative reactions on a range of chemically diverse substrates. Because of this, they are an excellent model system to understand how protein dynamics contributes to enzyme catalysis. In order to determine how the range of conformational subspace accessible to a model CYP enzyme effects ligand binding, in conjunction with our collaborators, we have performed 2D 1H,15N HSQC NMR chemical shift perturbation assays, fluorescence lifetime analysis, and x-ray crystallographic studies on the thermophilic cytochrome P450 enzyme, CYP119. Our results demonstrate that substrates and inhibitors bind to unique conformational substates through a process of conformational selection, not induced fit. Furthermore, these conformational substates are distinguishable on the HSQC NMR timescale. Fluorescence lifetime analysis suggests that the enzyme alternatively samples both an “open” and a “closed” conformation in the absence of ligand, and that large inhibitors preferentially stabilize the open conformation of the enzyme, while small inhibitors stabilize the closed conformation. X-ray crystallographic structures confirm these results. Together, these data allow us to propose a model of CYP ligand binding that may be useful for future design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts.

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Smita Mohanty










Title: Congenital Disorders of Glycosylation: Mechanisms of Oligosccharyl Transferase Function 
Smita Mohanty, Ph.D.
Associate Professor, Department of Chemistry
Oklahoma State University
Stillwater, OK

Oligosaccharyl transferase (OST) is a multi-subunit enzyme that catalyzes the co-translational Nglycosylation
of nascent polypeptides in the endoplasmic reticulum (ER). In the case of Saccharomyces cerevisiae, OST is composed of nine non-identical transmembrane protein subunits. In the central step of N-glycosylation, a preassembled oligosaccharide moiety is transferred to the asparagine side chain located in the Asn-X-Ser/Thr consensus sequence of the nascent polypeptides in the lumen of the endoplasmic reticulum (ER). Defects in N-glycosylation pathway can cause disorders known as congenital disorders of glycosylation (CDG). Complete loss of N-glycosylation is
lethal in organisms. Due to the inherent difficulties associated with integral membrane protein studies, the enzymatic mechanism of OST function remains obscure, although overwhelming results indicate the C-terminal domain of Stt3p subunit contains the acceptor substrate recognition and /or catalytic site. The structural and functional characterization of Ost4p subunit and the catalytic subunit of yeast OST, Stt3p will be presented.

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Robert Powers













Title: Metabolomics and drug discovery
Robert Powers, Ph.D.
Professor, Department of Chemistry
University of Nebraska-Lincoln
Lincoln, NE

Drug discovery is a complex and unpredictable endeavor with a high failure rate. Current trends in the pharmaceutical industry have exasperated these challenges and are contributing to the dramatic decline in productivity observed over the last decade. The industrialization of science by forcing the drug discovery process to adhere to assembly-line protocols is imposing unnecessary restrictions, such as short project time-lines. A diseased-centered systems biology approach to drug discovery provides a unique infrastructure to identify novel druggable targets and therapeutic agents to increase the efficiency and success rate of drug discovery. One important component of this approach is the use and development of NMR and MS based metabolomics techniques to monitor the in vivo activity and selectivity of potential drugs. Similarly, metabolomics can be used to monitor disease development, identify in vivo mechanisms of action for novel drugs, and evaluate mechanisms of drug resistance. Additionally, metabolomics may be an invaluable approach to easily and rapidly diagnose human disease and assist in personalized medicine by monitoring a patient’s response to a particular treatment. Our metabolomics technology, including our MVAPACK metabolomics software platform, PCA/PLS-DA utilities, and protocols for integrating NMR and MS data, sample preparation and metabolite identification will be discussed. Also, our analysis of the mechanism of action and resistance of TB and cancer drugs, and metabolic processes related to pancreatic cancer, Parkinson’s disease and a potential diagnostic tool for multiple sclerosis will be presented.           

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Susan Schroeder







Title: Structures, Dynamics, and Metal Ion Binding in Prohead RNA
Susan Schroeder, Ph.D.
Associate Professor, Department of Microbiology and Plant Biology
University of Oklahoma
Norman, OK

Prohead RNA (pRNA) is an essential component of a bacteriophage DNA packaging motor, but the role of pRNA in the mechanism of this bionanomotor remains a mystery.  Although the packaging motors in phi29 and GA1 bacteriophage have the same function, the sequence similarity of the ATPase and the pRNA are only 53% and 12%, respectively.  The structures, dynamics, and metal ion binding in the bulge loops of phi29 and GA1 prohead RNA have some surprising similarities and differences.   These comparisons can provide a structural basis for hypotheses about the role of prohead RNA in this bacteriophage nanomotor.

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