Second Winners of the SPECTROstar Nano Promotion:
Rising Stars in the Chemistry/Biochemistry Department at U.T. Arlington

Versatile Application of a SPECTROstar Nano System at U.T. Arlington

Kevin A. Schug, Frank W. Foss Jr., Roshan Perera, Kayunta Johnson‐Winters, Brad S. Pierce, Subhrangsu Mandal
Department of Chemistry & Biochemistry, The University of Texas at Arlington, Arlington, TX

 

Rising stars in the faculty of the Department of Chemistry & Biochemistry at the University of Texas at Arlington (UTA) request the placement of a SPECTROstar Nano plate reader instrument to develop and advance a myriad of high impact academic research and education initiatives. The goal of this proposal is to demonstrate a definite need for the innovative capabilities provided by this instrument in order to advance research in the areas of medicinal, analytical, physical, bioinorganic, and biological chemistry. The PIs represent a compilation of junior faculty who would benefit from, and disseminate the benefits of, an instrument such as the SPECTROstar Nano, which provides unparalleled capabilities, in terms of low‐volume high‐throughput UV/Vis spectroscopic measurements coupled with versatile assay functionality. Among other features, the proposed research will make use of the multiwell capacity and air‐sensitive sample handling ability of the instrument to carry out a myriad of kinetic and thermodynamic measurements. With our combined projects, the instrument will remain in constant use. Not only will it be an integral component to advance novel research avenues, it will be integrated into the educational curriculum of analytical chemistry, physical chemistry, and biochemistry laboratory courses. In this manner, virtually every undergraduate and many graduate students in the department (totaling close to 300 students at any given time) will gain hands‐on experience with this instrument. The students will no doubt carry this positive experience forward into their future scientific careers in academia, government, and industry.

The following lists short synopses of proposals designed by the faculty investigators requesting placement of a SPECTROstar Nano system at UTA. Use of the instrument will be supported in the proposed capacity by individual grant and start‐up funds, as well as by departmental resources. We would be happy to discuss the details of any and all of these projects in more detail, if desired.

Dr. Frank W. Foss, Jr. is an organic chemist interested in bioorganic and medicinal chemistry. He completed a Ph.D. from the University of Virginia and a post‐doctoral fellowship at Columbia University, before joining the faculty at UTA in 2007. His laboratory is establishing new methods for asymmetric catalysis and various medicinal chemistry projects related to combating infective agents.

HMP(P)‐Kinase is an essential enzyme for bacterial survival. The synthesis of bioactive 4‐amino‐5‐ hydroxymethyl‐2‐methylpyrimidine (HMP) analogues requires the efficient generation of in vitro kinetic and IC50 data. Among the numerous methods for measuring kinase activity, the SPECTROstar Nano would provide an efficient method for high‐throughput analysis. A major advantage over other methods would be the avoidance of chromatography, electrophoresis, or radiolabeling. The appropriate system would be capable of analyzing dozens of potential inhibitors with a number of assay replicates at one time over 6‐8 log orders of analyte concentration. HMP(K) activity can be investigated spectroscopically through an enzyme‐coupled experiment. The assay will detect the changing concentration of NADH+ (340 nm) to show the overall conversion of ATP to ADP. The speed and ease of data collection on a small scale from this instrument will greatly enhance this and other emerging medicinal chemistry projects in our laboratory.

The instrument will also aid the development of new catalytic reactions and reaction kinetics under aerobic and anaerobic atmospheres. Biomimetic analogues of riboflavin are attractive scaffolds for the development of small molecule catalytic oxidation reactions. Many flavin‐catalyzed reactions can be monitored by current spectroscopic methods; however, a major challenge to this research is the air sensitive nature of flavin analogues. The reduced flavin molecules react with oxygen. The ability to compare kinetic rates of reactions for multiple analogues under air, oxygen, mixed, and inert atmospheres with various terminal oxidants other than oxygen would be a significant advancement over our current technique for evaluating terminal (stoichiometric) oxidants to feed the catalytic reactions of interest. The SPECTROstar Nano offers the appropriate absorbance ranges for detecting arene substrate concentrations and a wide range of solvents, which will allow for reaction optimization. Furthermore, temperature control enables accurate kinetic studies for investigating mechanisms. Current methods of analysis require painstaking quenching of reactive oxygen intermediates and costly NMR or HPLC analysis. This instrument would allow efficient reaction optimization as well as mechanistic investigation through kinetic studies for the development of new biomimetic oxidation reactions.

Dr. Roshan Perera also joined the faculty at UTA in 2008, after receiving his PhD from the University of South Carolina and performing a post‐doctoral fellowship at the Scripps Institute. His recent efforts have led to the successful development of a new biosynthetic approach for functional protein arrays. The discovery of a covalent immobilization technique of functional proteins on solid surfaces (patent pending) was a significant step since this help overcome all the challenges that exist in this field.

The SPECTROstar Nano is an ideal system for testing the Perera lab's technique for homogeneous monolayer immobilization (covalent attachment) of functional proteins. This method is ready to be developed for high‐throughput assays using a microplate reader as the instrument of choice. Since the SPECTROstar Nano is capable of capturing ultra‐fast UV‐vis absorbance spectra in microLitre volume range (from 220 to 1000 nm in less than 1 sec/well), it would be interesting to see if we can immobilize and probe nanoMolar (~10 nM) concentrations of engineered proteins on the surface of the plate wells. As shown in Figure 1, we have demonstrated the homogeneous distribution of tagged protein on PEG derivatized glass chips by immobilizing a mutant enhanced green florescence protein (EGFP). We compared the florescence spectrum of EGFP protein in solution to the chip‐bound protein on the surface. The fluorescence spectrum of the chip‐bound mutant showed an emission intensity maximum at 510 nm (λex= 480 nm; λem= 500‐600 nm) which corresponds with about 10 nM free wild‐type EGFP in solution. An AFM topographical image shows that EGFP can cover an area of ~7‐15 nm2 as expected for a protein with diameter of ~ 3 nm and height of ~ 5 nm (PDB number 1Z1P). Once we have immobilized our proteins (we have the capability to engineer dozens of unique protein mutants with unnatural amino acids) in the well plates, Spectrostar Nano has the potential to capture the spectroscopic features of surface‐bound proteins, and their interactions with other proteins and ligands over a wide spectral range, to produce functional protein microarrays with broad applications in proteomics, diagnostics, biotechnology and life science fields.

Dr. Kayunta Johnson‐Winters recently joined the faculty at UTA in 2010. As a postdoctoral fellow at the University of Arizona (2007‐2009), she obtained the Ruth L. Kirschstein National Research Service Award from the National Institutes of Health.

Research interests in the Johnson‐Winters' laboratory are enzymes that use Cofactor F420. Cofactor F420 is an NAD(P) analog whose structure is reminiscent of 5‐deazaflavin. Our main interest is to study F420‐dependent Glucose‐6‐Phosphate Dehydrogenase (FGD) in a recombinant system. FGD is an essential enzyme found exclusively in Mycobacterium tuberculosis, the causative agent of TB and thus has great biomedical significance. Cofactor F420 enzymes have, in general, not yet been subject to rigorous enzymological investigation. Initial goals are to express and purify FGD and characterize the hydride transfer reaction mechanism of FGD and other Cofactor F420 dependent enzymes, using steady state and pre‐steady state kinetic methods. The kinetic activity of FGD is dependent upon cofactor F420. However, this cofactor is not synthesized in E. coli. Previous studies have revealed that the cofactor can be expressed from the nonpathogenic Mycobacteria smegmatis. The total yield of synthesized cofactor from Mycobacteria smegatis is low. Therefore, the use of the SPECTROstar Nano would be advantageous because very little volume is required for the UV‐Vis spectroscopy measurements. The use of this instrument would allow us to obtain an accurate concentration of the cofactor, without wasting precious material in the process. In addition, the SPECTROstar Nano spectophotometer would be useful for detecting cell growth of FGD and the detection of DNA concentrations of mutant FGD forms.

Dr. Subhrangsu S. Mandal joined the UTA faculty in 2005, and has been recently promoted to Associate Professor. The Mandal laboratory is funded by NIH, NSF, American Heart Association, and Texas‐ARP. His laboratory is highly focused on understanding epigenetic mechanisms of gene expression and steroid hormone signaling in humans. Additionally, in vitro and in vivo screening of synthetic and natural products in anti‐tumor and hormonal assays is performed to develop drugs for treating cardiovascular disease and cancer.

Access to a high throughput plate reader is crucial to the Mandal lab for performing cell‐based screening assays (MTT, luciferase‐based reporter, and mammalian hybrid assays, among others). Our goal is to understand the biochemical mechanisms of small molecule biological activities as they relate to molecular endocrinology, histone methylation, targeted gene therapy, and tumor growth inhibition. We have multiple patents pending and have published many scientific articles on this work, however, we are currently limited in our ability to address the libraries of compounds we have accumulated. Our proposal would be to make extensive utilization of the 1536‐well format to collect dose‐response and mechanistic data in a multitude of biochemical assay formats.

Dr. Mandal will also incorporate the SPECTROstar Nano into the biochemistry laboratory course curricula to teach students the value of spectroscopic based assay measurements.

Dr. Brad S. Pierce joined the faculty at UTA in 2008. Prior to this, he received his Ph.D. from Carnegie Mellon and he was an NIH postdoctoral fellow at the University of Wisconsin – Madison. Research in the Pierce lab utilizes a variety of biophysical and spectroscopic techniques (CD, UV, and EPR) to investigate the aspects of inorganic chemistry relevant to biologic chemistry and enzymology.

Dr. Pierce is interested in using the SPECTROstar Nano to develop a general strategy by which thiol dioxygenase (TDO) enzymes can be used as a template for the design of cheap, biomimetic synthetic catalysts for oxidative removal of organic‐sulfur from fossil fuel stocks. Oxidative desulfurization (ODS) represents an attractive green chemistry alternative to current hydrodesulfurization methods since the polar products of sulfur oxidation can be easily removed from the non‐polar fuels with minimal effort. One hallmark feature of all enzymes is their remarkable selectivity for their natural substrates over a wide range of structurally similar molecules. While evolutionally beneficial, this substrate specificity makes TDO enzymes less desirable as a model system for ODS catalysis. Therefore, a designed catalyst must remain highly specific for sulfur while also accommodating a variety of substrates.

Mutagenesis can be used to interrogate the role a particular amino acid plays during catalysis. For example,
substitution at a single amino acid position (R60A) decreases the specificity of CDO for its natural substrate by several orders of magnitude. However, each mutation must be specifically designed, implemented, and investigated one at a time; thus making this method impractical for simultaneously evaluating several amino acids or whole protein regions. Alternatively, by using an error‐prone polymerization chain reaction (ep PCR) procedure, we can generate a high frequency of random mutations localized specifically around the active site of the TDO enzyme. All of these mutants are then simultaneously transformed resulting in several hundred bacterial colonies, each with its own unique mutation which can be evaluated for thiol‐oxidation activity and substrate specificity.

As with any high‐throughput approach, the bottle neck in data collection resides in the ability to rapidly screen targets for a desired activity. Since it likely that the majority of mutants generated will result in a catalytically inactive enzyme, it is essential to develop a definitive method to differentiate between active and inactive TDO mutants. In these experiments, mutant clones generated by ep PCR will be transformed into commercially available BL21(DE3) E. coli for small‐scale overnight growth in the presence of thiol‐substrate surrogates and appropriate antibiotic for plasmid selection (37 °C). Moreover, the bacterial growth will be carried out in 96 well (1.3 mL/ea) bacterial plate using auto‐induction media. In principle, colonies expressing a viable TDO mutant will oxidize the thiol‐bearing substrates in the surrounding media. The presence of any unreacted thiols will be determined spectrophotometrically using Ellman's reagent which produces an intense yellow color at 412 nm upon reaction with free thiols. In these experiments, the SPECTROstar microplate reader will be crucial for rapid screening of enzymatic activity and relative substrate specificity before isolation of the expression plasmid for DNA sequencing.

Dr. Pierce also intends to incorporate the use of the platereader in the Biophysical Chemistry teaching laboratory to expose students to high throughput enzyme kinetics.

Dr. Kevin A. Schug joined the faculty at UTA in 2005 and has been recently promoted to Associate Professor. His research has a heavy focus on the development of high throughput binding determination methods using softionization mass spectrometry. Dr. Schug is funded by a CAREER award from NSF, among other grants, and was named the sole recipient of the 2010 Eli Lilly and Company Young Investigator Award in Analytical Chemistry.

Besides the design and implementation of new laboratory experiments for analytical chemistry curricula, Dr.
Schug will use the capabilities of the SPECTROstar Nano system to validate new methods for high throughput binding determinations based on flow injection analysis – mass spectrometry. It is well‐known that systematic errors in binding constants determined by mass spectrometry can arise due to the varying ionizability of different species in equilibrium. In order to avoid a major bottleneck in the evaluation of binding affinities for species taken from large compound libraries (natural products, aptamers, metallo‐organics, etc.), a complementary method is necessary to corroborate measured values. The large spectral range and well plate capacity of the instrument will provide unmatched versatility for interrogating a wide range of interaction systems. Furthermore, measurements in a microwell plate format will ensure that precious target compounds are needed only in small quantities to facilitate such measurements. In combination with mass spectrometric information, the combined data will lead to the ability to generate structure‐activity relationships for compounds against different targets (e.g. protein kinase enzymes). Given the potential for slow binding kinetics in this system, we will rely on the ability of the instrument to incubate and mix the protein‐ligand systems in an inert atmosphere prior to spectroscopic measurement.

In summary, we are confident that the proposed research and educational initiatives will make broad use of the innovative capabilities of this instrument. We will be pleased to publish our research in any or all formats, including application notes, conference proceeding, and peer‐reviewed journals to help promote this technology to the broader scientific community.

See the proposals of the other winners here.