The University of Arkansas is pleased to announce the formation of the Center for Protein Structure and Function.  The Center is supported by a five-year $9.63 million grant from the National Institutes of Health Centers of Biomedical Research Excellence (COBRE) program and by matching funds from the state of Arkansas.  The Center has nine senior and eight junior investigators from the Department of Chemistry & Biochemistry, Department of Biological Sciences and Department of Biochemistry and Molecular Biology.  Five new faculty will be hired in research areas which will complement the five project areas.  In addition, the Center will support twenty graduate research assistants and post-doctoral researchers.

    Protein structure and function is a central biomedical research area within structural biology, and is also crucial to the emerging field of structure-based drug discovery and design. With the human genome project nearing completion, protein structure/function determination is now considered to be a major opportunity for harnessing the wealth of new information about the genome to achieve improvements in human health. The University of Arkansas has made major commitments toward enhanced efforts in protein structure/function, by hiring new faculty, acquiring new instrumentation, and constructing two new Chemistry and Biology research buildings. The Center will build upon the unique research expertise of our faculty in the areas of X-ray crystallography, solution and solid-state NMR, mass spectrometry, computational chemistry, rapid laser-initiated kinetic analysis, peptide and drug design, and chemical synthesis. While building on current strengths, the establishment of the Center will substantially augment our capabilities in each of these areas, and will allow us to attract new colleagues to join our efforts. The unique combination of scientific expertise and state-of-the-art instrumentation in the Center will foster new opportunities for collaboration, and will position our faculty to develop innovative approaches to biomedical research problems.

    The need for a better understanding of protein structure and function can be seen in the vast increase of our knowledge of genomes. The last five years has seen an explosion of knowledge as the complete genomes of at least 24 organisms have been sequenced and published. The eagerly awaited results of the Human Genome Program will give us the sequences of the estimated 100,000 human genes. While we have already learned much from these projects, we have also learned the limits of our knowledge. The nucleotide sequence of any genome is readily translated into the amino acid sequence of the encoded proteins, but we know little about the structure and function of many of these proteins. The bacterium E. coli is the best characterized organism in the world, yet 38% of the 4288 proteins encoded in its genome have no known function. All five research projects within the Center will involve a multidisciplinary, collaborative approach to obtaining a better understanding of the structure and function of biomedically important proteins. Project 1 will focus on protein folding and orientation within membranes, and peptide transport via ABC-type permeases. Applications will include a better understanding of Clostridium infections and development of anti-Clostridium drugs. Project 2 will involve the development of new families of specific protein inhibitors as candidates for new drugs. Current targets include nucleic acid analogues that can bind to the NS3 helicase of the hepatitis C virus. Project 3 will explore new approaches to determining the structures of signal recognition particles, which facilitate protein targeting. Project 4 will focus on new experimental and theoretical approaches to understanding the principals governing protein folding. Project 5 will utilize powerful new rapid kinetics techniques to obtain a better understanding of energy coupling mechanisms in oxidative phosphorylation.

Dept. of Chemistry and Biochemistry, University of Arkansas
Dept. of Biological Sciences, University of Arkansas
Dept. of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences

Research Projects

Project 1: Peptide Transport and Interactions with Membranes and Extracellular Matrix

Project 2: Structure-based Drug Discovery

Project 3: Protein Targeting

Project 4: Principals of Protein Folding and Design

Project 5: Mechanism of Oxidative Phosphorylation

(Greathouse, Ivey, Sakon; Hinton, Koeppe, Schäfer).

    This project will concern the properties and biological applications of peptides that interact with membranes or with the extracellular matrix. Biological membranes and the extracellular matrix are barriers that define living cells, while at the same time providing communication pathways that link a single cell to others. New understanding of peptide/lipid interactions and peptide/matrix interactions will be sought. This information will be applied to develop potential new strategies for treating Clostridium infections. Two of our systems of investigation will be drawn from Clostridium, while a third fundamental approach will use a model system to explore peptide folding within a lipid environment. The specific aims are: 1. To understand peptide transport across membranes and the preferential use of low M_r peptides by Clostridium difficile for growth and colonization. 2. To elucidate general principles that govern the folding and biological function of membrane-spanning proteins. 3. To understand the mechanisms by which the extracellular matrix is recognized by a collagen-binding domain (CBD) from Clostridium histolyticum, and to develop peptides that could mimic the CBD. Such peptides would then be fused to autocrine/paracrine factors, such as growth factors, for the development of a wide range of novel therapeutic agents with potential clinical applications.

(McIntosh, Raney, Sakon, Turnbull, new hire; Schäfer).

    This project will develop and investigate small molecules for inhibition of specific protein functions. The design of these inhibitors and analysis of their effects on protein function will define how changes in protein structure relate to corresponding changes in function. This collaborative effort will draw on expertise in enzymology, chemistry, structural biology, and molecular modeling to design, prepare and assay these molecules. Compounds that show initial promise will be further optimized through combinatorial library development of more diverse structures. Our specific target proteins include helicase and tubulin. The two specific aims are:

I. Structure and Function Analysis of NS3 Helicase Through Inhibitor Development. The goal of this research is to design inhibitors for the Hepatitis C virus NS3 helicase using a rationally designed combinatorial approach. The research will also provide structure/function and kinetic mechanistic information on the helicase and its mode of translocation on single-stranded DNA.

II. Designed Ligands for Tubulin and Tubulin-binding Protein Cofactors.The goals of this project are to prepare anti-mitotic agents, and to develop ligands which inhibit tubulin assembly by binding to tubulin-binding cofactors or cofactor-tubulin complexes

(Henry, Sakon, new hire; Millett)

    Amino terminal signal sequences are hydrophobic targeting elements used to route proteins from the cytosol to the endoplasmic reticulum (ER). Signal sequences function in protein targeting through their interaction with signal recognition particle (SRP) in the cytosol. SRP binds the hydrophobic (H) core of signal sequences as they emerge from ribosomes. Once bound, SRP cotranslationally targets the entire ribosome-nascent chain complex (RNC) to the ER via its affinity for an SRP receptor at the ER. A 54 kDa subunit of SRP (SRP54) plays a central role in the targeting mechanism: SRP54 binds signal sequence H domains and binds the SRP receptor at the ER. An organellar SRP recently identified in chloroplasts (cpSRP) binds substrates by a novel post-translational mechanism, and therefore provides a unique model system to understand SRP/signal sequence interactions at the molecular level. The specific aims of this project will be to elucidate cpSRP54/H-domain interactions at the atomic level and to characterize structure/function relationships that enable cpSRP to uniquely bind substrates by a novel post-translational mechanism. This project will involve extensive collaboration between Ralph Henry, who is experienced in studying cpSRP-based targeting using molecular biology approaches, and Joshua Sakon, who is an experienced protein crystallographer.

(Stites, new hire; Hinton, Pulay, Wilkins)

    Our goal is to better understand the principles that underlie protein folding and structure. The vast increase of our knowledge of genomes needs to be matched by a corresponding increase in our understanding of the fundamental links between sequences and folded structures, and the ability to predict protein conformations. This collaborative project will involve the efforts of Drs. Stites (mutagenesis, crystallography), Wilkins (mass spectroscopy), Pulay (computational chemistry), and Hinton (NMR). The specific aims will be: 1. To examine critically the importance of hydrogen bonding and hydrogen bond networks for protein folding and conformational stability, including enhanced thermal stability. Both side-chain and main-chain/side-chain interactions will be examined using carefully designed mutations. 2. To examine critically the importance of hydrophobic packing interactions for protein folding and conformational stability, using a very extensive set of isoleucine, leucine and valine substitutions in the major hydrophobic core of staphylococcal nuclease. 3. To establish correlations between folding energetics and protein three-dimensional structure. This goal will be achieved examining at high resolution the structures of the mutant proteins by X-ray crystallography and NMR spectroscopy in solution. 4. To develop a new method for the prediction of the structural and energetic effects of packing mutations in proteins.

(Ivey, Stites, Davis, Durham, and Millett)

    Mitochondrial oxidative phosphorylation is a fundamental process in biological energy transformation, and disruption of this process leads to serious human health problems, including mitochondrial myopathies, degenerative diseases, and aging. However, it has not previously been possible to measure the actual rate of electron transfer between key redox centers in this process. We introduced a new method to initiate electron transfer by exciting a ruthenium complex with a nanosecond laser flash. The mechanism of energy coupling between cytochrome oxidase and ATP synthase is not well understood, particularly regarding the pathway of proton translocation. We propose a multidisciplinary, collaborative approach to this problem which combines powerful rapid kinetics techniques, bacterial model systems, and structure determination. The specific aims will be: 1. Carry out a detailed study of electron transfer in cytochrome oxidase from Bacillus firmus that combines rapid kinetics, site-directed mutagenesis, and structure determination. 2. Carry out a detailed study of proton translocation and ATP synthesis in the alkaliphilic B. firmus OF4 to assess the significance of alkaliphile-specific sequence motifs in the membrane-bound subunits of the F_O-ATPase. 3. Use NMR spectroscopy to study the structure of the membrane-bound subunits of the alkaliphile F_O-ATPase, primarily focusing on the isolated c subunit.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

....