Speakers

 

 

 

 

 

 

 

 

 

 

 

Plenary Speaker

John Markley
Steenbock Professor, Biomolecular Structure
Director:
National Magnetic Resonance Facility at Madison (NMRFAM) and BioMagResBank (BMRB)
Department of Biochemistry
University of Wisconsin-Madison
Madison, Wisconsin
Presentation Title: Conformational States and Interactions of Proteins Involved in the Biogenesis of Iron-Sulfur Proteins

Elaborate molecular machinery has evolved to assemble iron-sulfur clusters and to transfer them to apo-proteins as part of processes that presumably controls the toxicity of the Fe and S constituents. We present the results of our studies of this complex system in Escherichia coli cells and in human mitochondria. The machinery involves more than a dozen proteins that interact with one another in different ways to carry out the various steps involved.  We combine NMR spectroscopy, small angle X-ray scattering (SAXS), differential scanning calorimetry, isothermal titration calorimetry, assays of enzymatic activity, and other biochemical and biophysical approaches to determine the underlying mechanism of individual steps and to understand why defects in individual proteins are associated with disease or death. Several of the proteins adopt different conformational states depending on solution conditions or their interaction with other parts of the machinery.

Supported by U.S. National Institutes of Health grants U01GM94622; P41GM103399.

Justin Douglas

 

 

 

 

 

 

 

Justin Douglas
NMR Laboratory Director
University of Kansas
Lawrence, Kansas
Presentation Title: Automated structure verification in an academic synthetic chemistry workflow

The economic costs to the pharmaceutical industry of the development of therapeutic leads whose chemical structure has been determined incorrectly has motivated a systems of checks and controls built into the workflow for medicinal and analytical chemists.  Fueled by the increasing reliability of commercially available NMR spectral prediction algorithms, companies, from biotech to big Pharma, are incorporating Automated Structure Verification (ASV) of NMR data into corporate standard operating procedures.  The degree to which these tools can help minimize incorrect chemical structures in academic synthetic chemistry labs, which often rely on a less experienced work force, does not possess highly integrated IT and analytical support and does not possess top-down management hierarchy, is an open question.  This presentation serves as a progress report of our ongoing evaluation of ASV using 1D 1H and 13C NMR spectra recorded on compounds recently synthesized by several research groups at the University of Kansas.

Justin Douglas

 

John Laity
Associate Professor
School of Biological Sciences
Division of Cell Biology and Biophysics
University of Missouri at Kansas City
Kansas City, Missouri
Presentation Title: Probing the Functional Roles of MTF-1 and p300 in Mammalian Zn(II) Homeostasis: NMR and Calorimetric Studies Implicate Zn(II) Binding Heterogeneity in the p300-TAZ2 and MTF-1 DNA Binding Domains as Key Determinants

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Katie Mitchell-Koch
Assistant Professor
Department of Chemistry
Wichita State University
Wichita, Kansas
Presentation Title: Connections between electronic structure, protein environment, and fluorine chemical shifts

Computational methods, primarily DFT calculations, have been used to understand the 19F chemical shifts of fluorinated amino acids.  Fluorine nuclei are known to be exquisitely sensitive to changes in environment, and can serve as reporters on biological and chemical events, such as protonation, changes in dielectric environments, ligand binding, and protein folding/unfolding.  However, the origins of the changes in fluorine chemical shifts are poorly understood, and shielding/deshielding does not always follow the well-established patterns known for other nuclei, especially 1H.  Our work on 2-fluorohistidine and 4-fluorohistidine have shown connections between fluorine charge, C-F bond polarity, and 19F chemical shifts, but the calculated 4-fluorohistidine spectra exhibit abnormal or “reverse” behavior.  This appears to be a consequence of delocalized fluorine molecular orbitals.  Work is underway with other systems known to exhibit normal and reverse behavior, to see what correlations can be made between electronic structure, molecular structure, and fluorine chemical shifts.

Steven R. Van Doren

 

 

 

 

 

 

 

 

 

Steven R. Van Doren
Professor
Department of Biochemistry
University of Missouri at Columbia
Columbia, Missouri
Presentation Title: Transient interactions of Metalloproteinases with Bilayers by Paramagnetic NMR

Matrix metalloproteinases-7, -12, and -14 are secreted by macrophages. They play beneficial antimicrobial roles, as well as checkered roles in cancer, arthritis, and cardiovascular disease. Though MMP-7 and -12 are water-soluble and can diffuse in the extracellular matrix, we find both proteases to have dual modes of binding bilayers, using paramagnetic NMR with mobile labels and fluorescence with fixed labels. Restrained molecular dynamics using fluid bilayers helps in visualizing these interactions.  Both MMPs are quickly internalized via membranes, with MMP-12 consistently entering the nucleus.  MMP-14 or MT1-MMP instead is tethered to cell membranes via a transmembrane helix. Without this anchor, the soluble C-terminal domain still associates with membrane mimics, according to the NMR and fluorescence assays.  Biomedical implications of the membrane interactions will be mentioned.

Supported by NIH grant GM057289, supplemented by CA098799.

 

 

 

 

 

 

 

 

Vincenzo Venditti
Assistant Professor
Department of Chemistry
Iowa State University
Ames, Iowa
Presentation Title: Versatile dynamics in the C-terminal domain of bacterial Enzyme I

Enzyme I (EI) is a multidomain, 128-kDa homodimer that exists in two conformational states related to one another by two large (50–90°) rigid body domain reorientations. The open conformation of apo EI allows phosphoryl transfer from His-189 located in the N-ter domain (EIN) to the downstream protein partner. The closed conformation brings the EIN domain into close proximity to the C-ter domain (EIC), thereby permitting in-line phosphoryl transfer from phosphoenolpyruvate bound to EIC to His-189. Here, by using NMR, small-angle X-ray scattering, enzyme kinetic assays and computational methods we show that the EIC domain undergoes an expanded-to-compact conformational equilibrium on the μs-ms that regulates ligand-binding to EIC, and plays a crucial role in stabilization of the catalytic transition state. Finally, we show that complete suppression of EIC dynamics upon substrate-binding promotes the open-to-close conformational switch and autophosphorylation of full-length EI.

Liliya Vugmeyster

 

 

 

 

 

 

 

 

 

Liliya Vugmeyster
Assistant Professor
Department of Chemistry
University of Colorado-Denver
Denver, Colorado
Presentation Title: Dynamics and solvation of amyloid Abeta

Amyloid fibril deposits found in Alzheimer’s disease patients are comprised of Amyloid-b (Ab) protein forming a number of hydrophobic interfaces which are believed to be mostly rigid.  We have investigated the ms–ms time scale dynamics of the intra-strand hydrophobic core and interfaces of the fibrils comprised of Ab1-40 protein. Using solid-state 2H NMR line shape experiments performed on selectively deuterated methyl groups, we probed different polymorphs of native Ab as well as the protofibrils of D23N Iowa mutant, associated with an early onset of Alzheimer’s disease. Surprisingly, all sites and polymorphs exhibited significant flexibility with motions persisting down to at least 200 K. Modeling and comparison with other globular proteins indicate that the driving force behind this effect is due to kinetics, specifically, low activation energy barriers for rotameric jumps. The results on the hydration dependence support the existence of water-accessible cavity recently predicted by MD simulations.

   

 

 

 

 

 

 

 

 

 

Erik R.P. Zuiderweg
Professor
Department of Biological Chemistry
University of Michigan Medical School
Ann Arbor, Michigan
Presentation Title: Diffusive and tethered complexes by NMR

Due to selective determination and publication, protein-protein and protein-ligand complexes are often considered to be well-defined structures. However, many complexes and especially encounter complexes are much less-well defined, and remain remarkably dynamic by surface diffusion, tethering or a combination. It appears that such complexes are dominated by electreostatic interactions. NMR is well-suited to describe such complexes and several examples, mainly in the area of Hsp70 chaperones will be presented.