Symposium Speakers

Rafael BruschweilerRafael Bruschweiler

 

 

 

 

 

Plenary Speaker

Rafael Brüschweiler

Professor
Ohio Research Scholar
Department of Chemistry and Biochemistry
Ohio State University
Columbus, OH
Advanced complex mixture analysis by NMR and NMR/MS for metabolomics

The rapid, reliable and comprehensive identification and quantitation of a large number of organic molecules in complex mixtures, such as metabolites in biological systems in the context of metabolomics, will be discussed using multidimensional NMR tools. They include curated databases of known metabolites, multidimensional spectral query for identification and quantitation, and rapid non-uniformly sampled 2D 1H-1H TOCSY collection and spectral reconstruction. Many of these developments are well-suited for automation and have been integrated into our COLMAR suite of web servers and databases (http://spin.ccic.ohio-state.edu/index.php/colmar). Since for most biological samples a large number of spectral features belong to unknown metabolites, there is a pressing need to identify them accurately and efficiently. An approach will be described that synergistically combines multiple sources of information, including NMR, cheminformatics, and/or high-resolution mass spectrometry.

Elan Eisenmesser

Elan Eisenmesser

 

 

 

 

 

 

 

 

Invited Speakers

Elan Eisenmesser

Associate Professor, Molecular Biology Program
School of Medicine
University of Colorado Anschutz Campus
Aurora, CO
Protein dynamics drives redox regulation.

Our main interests are to understand how dynamics within both the active sites of enzymes and distally coupled dynamic networks modulate function.  Human Biliverdin Reductase B (BLVRB) has recently emerged as a critical redox regulator that can control hematopoietic cell fate and we have shown that human BLVRB is highly abundant in red blood cells that are in turn the most highly abundant cell in the human body. However, little is know in regard to how this ubiquitously expressed enzyme functions, much less the role of dynamics in function. Our NMR studies have begun to pinpoint how critical residues within the BLVRB active site “clamp” over its coenzyme and how distally coupled motions modulate coenzyme binding. Evolutionarily guided studies using multiple BLVRB homologues have revealed a surprising swap of residues important for “clamping” and identified links between conformational sampling and coenzyme binding for BLVRB family members. Collectively, our studies clearly indicate the importance of inherent enzyme movements within the BLVRB family and enzyme function.

Nathan Olyer

Nathan Oyler

 

 

 

 

 

 

Nathan Oyler

Associate Professor
Department of Chemistry
University of Missouri-Kansas City
Kansas City, MO
Using NMR to Quantify Drug Release from Pharmaceutical Formulations

Accurately assaying the amount of drug released (as a function of time) in human bodily fluids is a crucial requirement of the drug formulation development process (i.e. developing the matrix to enclose the drug).  Classically, a method combining dialysis (to separate the released drug from the bodily fluid) and HPLC (to quantify the amount of drug) is commonly used due, in part, to its high sensitivity, but here we demonstrate a more direct and accurate method to determine the real-time release using quantitative NMR.  The fundamental NMR concept that makes the method possible relies on the difference in rotational correlation times between the bound drug and free drug.  We also demonstrate the pitfalls of using the dialysis/HPLC method as well as demonstrate directly detected real-time drug release profiles of drug formulations dissolved in various simulated bodily fluids.  Additionally, the extensibility of the method to other drugs/formulations will be discussed.

Om Prakash

Om Prakash

 

 

 

 

 

 

Om Prakash

Professor, Department of Biochemistry and Molecular Biophysics
Director, NMR Facility
Kansas State University
Manhattan, KS
NMR Studies of a Novel Family of Neutrophil Serine Proteases (NSP) Inhibitors: Toward Understandingthe Structural Basis for Complement Inhibition by Staphylococcus aureus Innate Immune Evaision Proteins

Staphylococcus aureus secretes an array of proteins, many of which serve to disrupt the host’s innate immune system from recognizing and clearing bacteria with optimal efficiency. Our collaborator Dr. Geisbrecht’s laboratory has identified a novel group of secreted staphylococcus proteins that inhibit the function of neutrophil serine proteases. One such family of evasion proteins, called extracellular adherence protein (Eap) is a multidomain protein that participates in various protein-protein interactions that inhibit the innate immune response, including both the complement system and Neutrophil Serine Proteases. Specifically, Eap binds with nanomolar affinity to complement protein C4b, and thereby blocks binding of the classical (CP) and lectin pathway (LP) pro-protease C2 to C4b. This effectively eliminates formation of the CP/LP C3 proconvertase, which is required for amplification of downstream complement activity and subsequent inflammatory events. To provide a structural basis for understanding the C4b-binding properties and complement inhibitory activity of Eap domain proteins, we have initiated the NMR structural studies on these proteins.

Julien RocheJulien Roche

 

 

 

 

 

Julien Roche

Assistant Professor
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology
Iowa State University
Ames, IA
From IDP to IDR in MFP

I will present a series of NMR methodological approaches for the characterization of intrinsically disordered proteins, amyloid peptides and mixed folded proteins containing long disordered regions. I will discuss the use of high-pressure NMR to explore the structural properties of a-synuclein. I will also show how large sets of NMR parameters, including chemical shifts, NOEs, and J-couplings (3JHNHa, 3JCʹCʹ, 3JCHa, 1JHaCa, 2JCaN and 1JCaN) can be combined to provide a detailed structural characterization of the amyloid peptides Ab40 and Ab42. New NMR experiments were specifically designed to measure J-couplings and NOEs with the highest possible precision. I will finally present the wide range of biophysical techniques required for the characterization of DISC1, a long scaffold protein involved in schizophrenia and bipolar disorders, containing both structured and disordered regions.

Beat Vogeli

 

 

 

 

 

 

 

 

 

Beat Vogeli

Assistant Professor
School of Medicine, Department of Biochemistry and Molecular Genetics
University of Colorado Anschutz Campus
Aurora, CO
Sub-ångström resolution in RNA, protein disorder in dynein-dependent cargo transport

As a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely obtained from NOE data alone. Instead, additional semi-empirical restraints and labor-intensive techniques are required for structural averages, while there are only a few experimentally derived ensembles representing dynamics. We show that our exact NOE (eNOE) based structure-determination protocol is able to define a 14-mer UUCG tetraloop structure at high resolution without other restraints. All animal cells use the motor cytoplasmic dynein 1 to transport diverse cargo towards microtubule minus ends and to organize microtubule arrays. Cargo-specific adaptors engage with dynein to recruit and activate the motor. Using NMR, we demonstrate that the C-terminal region of human dynein light intermediate chain (LIC-C) is intrinsically disordered but contains two short conserved segments with helical propensity, which are the binding sites for various adaptors. We confirm our identified key residues by mutations in C. elegans LIC-C, which severely affect development, locomotion, and life span of the animals.