INBRE Faculty Associates in Structural Biology

CREIGHTON UNIVERSITY COLLEGE OF ARTS AND SCIENCES

"Understanding Genetic Regulation Through Structural Studies of Riboswitch-Metabolite Complexes"
Juliane K. Soukup, Ph.D.
Collaborators: Gloria Borgstahl, Ph.D. (UNMC) and Rob T. Batey, Ph.D. (University of Colorado at Boulder)

Abstract: The emergence of antibiotic resistance has required that new approaches be applied in order to effectively fight a host of medically relevant bacterial infections. The currently used, imprecise antibiotics need to be replaced with novel, rigorous, and safe treatments in order to combat the evolved bacterium of today. One way to destroy bacteria is to target one of their most essential processes, metabolism. The discovery of RNA structural elements, termed riboswitches, that bind cellular metabolites and control expression of essential metabolic genes provides a unique and distinct target for development of artificial agonists to fight bacterial infections.

In order to rationally design and develop effective artificial agonists/antibiotics that target bacterial riboswitches, an understanding of the structural and functional details of the riboswitch-metabolite complex is essential. The aims of this grant focus on (1) probing the molecular contribution of a conserved base pair distal to the metabolite binding site within a riboswitch that binds guanine, (2) designing a crystallization construct for structural characterization of the newly discovered pre-queuosine1 riboswitch, and (3) determining the molecular interactions between the metabolite pre-queuosine1 and its riboswitch aptamer domain. The studies described here will provide atomic level detail of the interactions between riboswitches and their ligands. Structural studies of riboswitches are essential in order to gain detailed information about how the RNA interacts with its metabolite and to ultimately design non-natural metabolite analogs that can act as antibiotics. The X-ray crystallography studies described here will also have a high impact on understanding RNA-based gene regulation.

"Assessment of Cellular Energetics by NADH FLIM"
Michael G. Nichols, Ph.D.
Collaborator: Richard Hallworth, Ph.D. (CUMC)

Abstract: Metabolic homeostasis, or the ability to closely match energy production with demand, is a fundamental requirement for the health and normal functioning of the cells, tissues, and organs of the body. Therefore, several technologies have been developed to assess cellular energy production. Optical imaging modalities are particularly attractive, because light is minimally invasive, easily delivered to the various tissues of the body, and capable of providing rapid feedback with high spatial resolution. One approach to characterizing the metabolic state has been to use the intrinsic fluorescence emission from the reduced form of nicotinamide adenine dinucleotide (NADH) and flavoproteins that directly participate in mitochondrial energy production. This has been extensively applied to a variety of tissues and has been instrumental in the development of our current understanding of the regulation and maintenance of the metabolic state. However, interpreting the observed changes in tissue fluorescence can be problematic, often requiring assumptions or the need of additional measurements for effective application. Traditionally, metabolic imaging has employed fluorescence intensity as a surrogate for the concentration of electron donors to the respiratory chain. Recent studies, however, have shown that these measurements of fluorophore concentration may be error prone. Fundamentally, this is because the fluorescence intensity is dependent on the local environment of the fluorophore and is properly expressed as a product of both the fluorophore's lifetime and concentration. Since lifetime changes can also occur as a result of changes in the ratio of the free to enzyme-bound fluorophores populations, changes in intensity are difficult to interpret. Other studies have suggested that NADH fluorescence lifetime imaging (FLIM) may provide a more accurate measurement of cellular energetics. While FLIM is a promising, novel alternative, it has yet to be properly evaluated in well controlled, yet realistic cellular environments. Using easily manipulated yet relevant in vitro cultures, we propose to systematically compare measurements of the cellular metabolic state obtained from NADH FLIM with the traditional assessment made using fluorescence intensity alone. By establishing the advantages and limitations of this new technique, we will be able to properly deploy metabolic imaging techniques to better characterize, diagnose and develop treatment interventions for a broad range of human disease.

WAYNE STATE COLLEGE

"Folding of Recombinant Major Outer Membrane Protein cysteine Mutants from Chlamydia: Implications for Vaccine Development"
Gustavo Zardeneta, Ph.D.
Collaborator: Luis de la Maza, Ph.D. (University of California at Irvine) and Jose Mendoza, Ph.D. (California State University at San Marcos)

Abstract:  The chlamydial envelope like that of other Gram negative bacteria has an inner and outer membrane (OM) separated by a periplasm layer, which appears to be devoid of peptidoglycan. Cysteine-rich proteins, such as the 60 kDa OmcB, 12 kDa OmcA and MOMP are believed to form a disulfide network that provides rigidity to the outer membrane. The chlamydial MOMP belongs to a family of transmembrane proteins with porin activity and similar molecular structure: homotrimers where each 39 kDa monomer forms a β barrel structure. However, chamydial MOMPs have more cysteines and disulfide bonds per monomer than other porins from this superfamily. The MOMP from the Chlamydia trachomatis mouse pneumonitis (MoPn) serovar, which will be used in this proposal has 8 cysteines: 4 involved in disulfide bonds and 4 present in a reduced state in purified nMOMP [18]. It is not known if the reduced residues are involved in intermolecular bonds in vivo.  The role of disulfide bonds in the MoPn MOMP's folding, insertion into the OM, and protein stability is not known although it has been shown that these bonds are not necessary for maintaining the trimeric structure in vitro [15]. All known porins form trimers without disulfide bonds. To decipher the in vivo role of this novel molecular structure and conserved residues, it is paramount to understand how the disulfide bonds affect the protein's ability to fold in vitro and their role in the MOMP's stability. Several outer membrane proteins have been folded with ease using detergents or lipid-containing mixed micelles (reviewed in [9]), without the need for redox control. Although the native MoPn MOMP has yet to be refolded using similar strategies, ostensibly due to its high cysteine content and disulfide bonds, the author has partially-unfolded native MOMP to a monomer and correctly refolded the monomeric protein to its native form as judged by its reactivity towards conformational antibodies and by protein electrophoresis [Zardeneta et al., submitted for publication]. The latter techniques allows for a quick evaluation of refolded MOMP since only the correctly folded MOMP migrates as a trimer, rather than a monomer, in SDS-PAGE, provided the protein sample is not heated prior to loading onto gel. The goals of this proposal are to unfold/ refold recombinant MOMPs, where cysteines involved in disulfide bonds are systematically replaced by serine by site directed mutagenesis. Results from this study will be a starting point for future biophysical analyses aimed at understanding the thermodynamic and kinetic stability, assembly mechanisms and incorporation of the MOMP into the bacterial outer membrane.

DOANE COLLEGE

"Optically Enhanced Oligonucleotides to Screen for Metabolic Diseases"
Andrea E. Holmes, Ph.D.
Collaborator: Garrett A. Soukup, Ph.D. (CUMC)

Abstract: Current screening methods for metabolic diseases are too costly, expensive, and time-consuming for broadly testing all newborns. Thus, many metabolic diseases go undetected and untreated for many years and screening is implemented only for the one most common metabolic disease. Using the proposed sol-gel supported aptamer-dye mix-and-measure assays in urine allows for cost-effective mass screening. The proposed research is dedicated to the development of new, highly selective, and sensitive molecular sensors that change color or fluorescence properties in the presence of steroid metabolites. Several steroids are disease markers for inborn metabolic diseases. We are interested in mapping three-way junctions of oligonucleotide-based binders (aptamers) to determine general principles that can be used in understanding molecular recognition of fluorescent dyes, visible dyes, steroids, and their metabolites by aptamers. The screening of aptamer-dye complexes will address an important medical need by generating cost-effective, rapid, mix-and-measure methods suitable for mass screening and monitoring of steroids or metabolites caused by faulty steroid metabolism. Organic dyes that form strong complexes with binding aptamers and metabolites will be found using a combinatorial 96-well plate approach. Reliable aptamer binding assays by UV-Vis and fluorescence spectroscopy have been established by our group. Furthermore, aptamers will be incorporated into colloidal silicate polymers for the development of handheld solid sensor devices to be used in medicinal applications. "Sol-gels" are based on solid colloidal silicates that will form a porous hard gel and will incorporate the aptamer-dye complexes within the gel, allowing for attaching the new molecular sensors into a solid support.

"Roles and Mechanisms of Non-collagenous Proteins in Biomineralization"
Erin E. Wilson, Ph.D.
Collaborator: Gerard S. Harbison (UNL)

Abstract: Mineralized tissues are composites of organic (collagen, other proteins) and inorganic (mineral) components. Biomineralization of these tissues is closely controlled by non-collagenous proteins (NCPs) within the tissue organic matrix to produce biological materials with unique mechanical and biological properties tailored for their functions. The size, shape, orientation and location of mineral crystals is regulated by these proteins in order to achieve these remarkable properties. Control of biomineralization is incompletely understood, including the NCPs involved in the regulation in different tissues and their specific roles, changes that occur in diseases involving mineralization, and the physicochemical mechanisms employed by NCPs to control mineralization through direct NCP-mineral interactions. The proposed research aims to elucidate the molecular events of biomineralization through two project components. The first component is the development of a gel electrophoresis/diffusion system to facilitate fast analysis of collagen-based tissues for these mineralization regulators. The proposed gel system will be capable of separating small amounts of mineralization-regulating species from complex tissue matrices and evaluating their effect on mineralization in vitro. Once optimized, this gel system will be used to evaluate differences in NCP complements at different stages of mineralization, as well as in healthy versus diseased mineralized tissue. The second component of the project aims to investigate structure and conformation changes in NCPs at the interface of protein and mineral. Two structural techniques, circular dichroism (CD) spectrophotometry and solid-state nuclear magnetic resonance (NMR) will be employed to investigate the structure of NCP-derived peptides adsorbed to mineral crystals. From these investigations a model of the protein-mineral interaction will emerge, shedding light on the physicochemical mechanisms by which these proteins influence mineral growth in vivo.

Mineralized tissues are composites of organic (collagen, other proteins) and inorganic (mineral) components. Biomineralization of these tissues is closely controlled by non-collagenous proteins (NCPs) within the tissue organic matrix to produce biological materials with unique mechanical and biological properties tailored for their functions. The size, shape, orientation and location of mineral crystals is regulated by these proteins in order to achieve these remarkable properties. Control of biomineralization is incompletely understood, including the NCPs involved in the regulation in different tissues and their specific roles, changes that occur in diseases involving mineralization, and the physicochemical mechanisms employed by NCPs to control mineralization through direct NCP-mineral interactions. The proposed research aims to elucidate the molecular events of biomineralization through two project components. The first component is the development of a gel electrophoresis/diffusion system to facilitate fast analysis of collagen-based tissues for these mineralization regulators. The proposed gel system will be capable of separating small amounts of mineralization-regulating species from complex tissue matrices and evaluating their effect on mineralization in vitro. Once optimized, this gel system will be used to evaluate differences in NCP complements at different stages of mineralization, as well as in healthy versus diseased mineralized tissue. The second component of the project aims to investigate structure and conformation changes in NCPs at the interface of protein and mineral. Two structural techniques, circular dichroism (CD) spectrophotometry and solid-state nuclear magnetic resonance (NMR) will be employed to investigate the structure of NCP-derived peptides adsorbed to mineral crystals. From these investigations a model of the protein-mineral interaction will emerge, shedding light on the physicochemical mechanisms by which these proteins influence mineral growth in vivo.

UNIVERSITY OF NEBRASKA OMAHA - IST&E

"An Innovative Technique for Classification of Fungal Sequences using Restriction Enzyme Cut Order"
Hesham H. Ali, Ph.D.

Abstract: Restriction fragment length polymorphism (RFLP) of chromosomal DNA is one of the powerful tools used in the fingerprinting of microorganism and various other molecular and epidemiological studies. In the laboratory, the standard approach allowing pattern-based classification of organism using RFLP begins with the digestion of DNA with one to two restriction enzymes, which is followed by gel electrophoresis. This laboratory approach may not be practical when experimental data set include large number of genetic sequences. However, computational approach can augment the process of choosing an ideal set of restriction enzyme. Additionally, the RFLP process can be simulated in-silico where computation intensive sequence alignment methods are currently being used. In this study we introduce a novel concept of Enzyme Cut Ordera restriction enzyme based characteristics of DNA sequences which can be defined and analyzed computationally. A similarity matrix is developed based on the pairwise Longest Common Subsequences (LCS) of the Enzyme Cut Orders, where the set of enzymes used for the analysis is obtained by using genetic algorithm and the target sequences used include the internal transcribed spacer regions of rDNA from fungi. Unlike alignment-based methods, the proposed approach is alignment free and the enzyme cut order is used throughout the computational process including sequence comparison, clustering and identification. The preliminary results obtained from this approach show that the organisms that are phylogenetically closely related forms a single cluster and successful grouping of phylogenetically close or distant organism is dependent on the choice of restriction enzyme used in the analysis. This novel alignment-free method, which utilizes the Enzyme Cut Order and restriction enzyme profile, is a reliable alternative to the local or global alignment based classification of organism.

"Alignment-free Approach in Genomic Sequence Comparison"
Dhundy R. Bastola, Ph.D.
Collaborator: Hesham H. Ali, Ph.D. (UNO-IST&E)

Abstract: The massive amount of data inundating life scientists is a common denominator driving various bioinformatics research activities. There are many interesting problems in life sciences whose solutions are hidden in these data and there are many potential approaches in the engineering and computer science domain to finding solutions to these problems. Dr. Bastola's research is focused on computational tool development and deployment in bioinformatics. Traditionally we have used single target genes (rRNA for ribosomal small or large subunits) for developing rapid identification assays and studying evolutionary mechanisms in living organisms. Computational tools like the BLAST developed for such studies employ alignment-based pair-wise sequence comparison. Today we have access to genomic sequences for a number of organisms, and biologically meaningful information can be obtained from comparative genomic analysis of the whole genome. Alignment-based computational tools cannot handle genome comparison and represent a serious limitation and hindrance to the discoveries that are possible with comparative genomic analysis. The proposed study will evaluate an alignment-free approach to genomic sequence comparison, where information content in the DNA is compared instead of the nucleotide sequence. This approach requires us to visualize both DNA encoding and computer programming to follow a simple linear progression from information to function: DNA to protein in biology, and High Level Language to executable machine instructions in computers.

"Ontology Development and Database Annotation for Influenza Informatics"
Zhengxin Chen, Ph.D.

Abstract: The objective of this research proposal is to develop a prediction system of mutations and genotypes for influenza surveillance and vaccine strain selection. The specific hypothesis behind this proposed research is that evolution and pathogenesis of influenza A viruses are ultimately determined by the information embedded in the genomes. The rationale underlying the proposed research is that, once genetic mutations linked to the evolutionary lineages, host specificity, and the level of pathogenicity have been uncovered, the most significant mutation sites can be used to trace or predict evolutionary changes of newly emerging influenza A viruses and to develop effective vaccines so that influenza outbreaks can be prevented and controlled. In addition to the supportive data (see Preliminary Studies), we are particularly well prepared to undertake this research because the project will capitalize on our institution's proven commitment to promoting bioinformatics research and education. In addition, our university is committed to sustaining this program after the requested funding terminates.

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