(Timm Knoerzer, Ph.D. in collaboration with Prof. José Luís Mascareñas, Universidad de Santiago de Compostela, Spain)
Purpose of the project/Problem to be solved:
To synthesize and characterize a protein-functionalized tetrameric molecule capable of binding selectively to specific promoter regions in DNA and which may hold the capacity for future development into a switchable system which can be turned on or off using differential chemical signals/cues.
Background and Introduction:
DNA is the molecule that stores genetic information in almost all forms of life. Following the
processes of transcription and translation, this information is expressed in the form of proteins which carry out various cellular functions. However, the ultimate site of regulation for the expression of these proteins exists at the level of transcription. The essential product of transcription is a second messenger molecule called mRNA. The synthesis of mRNA is catalyzed by an enzyme called RNA polymerase II, which only becomes active upon interaction with certain proteins called transcription factors. Normally, these transcription factors lie dormant in the cell, but become activated upon interaction with other signaling mechanisms (e.g. via the interaction with some ligand). This activation of the transcription factor typically induces a change in the conformation of the protein that then affords an extremely specific interaction with a recognition sequence near the 5’ end the DNA sequence.
The site specificity of this interaction is remarkable given that an individual transcription factor will recognize some small sequence of base pairs among millions that exist in the genome.
Statement of proposed project:
Our research is focused on the bZIP family of transcription factors, most notably GCN4. The
objective is to design and synthesize miniature peptidomimetic molecules that retain the capacity to bind to DNA with great specificity.
Michelle Scotland: Fate of Dipicolinic Acid Released from Bacillus subtilis During Spore Germination
Bacillus subtilis (B. subtilis) is a gram positive bacterium that forms metabolically dormant
spores under starvation conditions. The nonpathogenic bacterium is a model organism for other spore-forming bacteria that cause disease such as anthrax, gangrene, botulism, and food
poisoning. Spores of B. subtilis are resistant to a variety of environmental stresses and will remain dormant until nutrients become available. The spore has a unique structure with several layers including the coat, outer spore membrane, cortex, germ cell wall, and inner spore membrane (Figure 1). At the center of the spore is the core which contains nucleic acid, tRNAs, proteins and a small molecule known as dipicolinic acid (DPA), which is chelated to Ca2+ (Figures 1 and 2). During sporulation DPA replaces water in the core and functions in providing spore resistance to wet heat and UV radiation. When nutrients become available, the spore undergoes germination, a process that converts the spore back into a vegetatively growing cell.
Although it has been shown that DPA is released from the spore’s core at the start of
germination, the fate of DPA after release is unknown. In our research we are investigating
whether or not B. subtilis and/or another microorganism can use DPA as a nutrient source.
(Timm Knoerzer, Ph.D. in collaboration with Prof. Benjamin Miller, University of Rochester Medical Center)
The primary aim of this proposal is to explore the utility of the phenol-amine hydrogen bond as a fundamental structural element, and as an organizing principle for molecular recognition. A detailed understanding of the individual covalent and non-covalent interactions that contribute to the production of a stable molecular conformation is a critical prerequisite to our ability to predict and manipulate molecular structure and function. The Miller Group at the University of Rochester recently discovered a synthetic receptor built on a tyrosine-functionalized cyclohexane core that organizes itself into a folded conformation based on hydrogen bonding between phenolic group and neighboring amine groups (J. Am. Chem. Soc. 2006, 128(8), 2532-2533. DOI: 10.1021/ja058126p). While the phenol-amine H-bond is a known structural motif, it has received only limited study in solution. In order to better understand the properties of the phenol-amine Hbond, we propose the synthesis of six analogs in which the phenol pKa, and therefore its propensity to hydrogen bond, will be systematically varied. NMR experiments (T1 and T2 relaxation measurements and 2-dimensional NOESY spectra) may be carried out as a function of pH to assess the formation and stability of H-bonded structures. Similarly, the ability of these receptors to bind anions and cations as a function of pH may be assessed by isothermal titration calorimetry (ITC). Second, our method may be extended to pKa determination by ITC to amines, allowing us to probe changes in the amine pKa as a function of hydrogen bonding strength, and providing a general method for the study of amine pKa in other systems.
Lauren Wright: Isolation of lipolytic soil microbes and quantification of lipase-mediated lipid degradation
The lipolytic potential of soil microbes was investigated to identify bacterial strains with
an exceptional ability to hydrolyze the lipids of nonvirgin soybean oil. Nonvirgin oil may
be obtained easily from restaurants and is the most economical choice for use in biodiesel
production. Most commonly, a base-catalyzed transesterification process is used to convert oil to biodiesel. Nonvirgin oils, however, due to their high concentration of free fatty acids, produce soap as an undesirable byproduct of the transesterification reaction. To eliminate soap formation, and therefore increase the efficiency of biodiesel production, nonvirgin oil can be degraded, or broken down, into a free fatty acid feedstock; the feedstock may be converted to biodiesel by an acid catalyzed esterification process, which results in no soap formation and greater product-yield. The goal of the present experiment was to identify lipolytic, or lipase-secreting, soil microbes that quickly break down the triglycerides of soybean oil into free fatty acids. Soil samples from the oil storage sites of local restaurants were collected and cultured to isolate pure bacterial strains. Isolated bacterial colonies were grown on tributyrin-rich media as a test for lipolytic potential. Cultures that secreted lipase were subjected to a regular lipase
activity assay to determine the concentration of released enzyme. A free fatty acid determination test was performed using soybean oil of known fatty acid concentration to quantify the lipolytic ability of each bacterial strain.
Many low quality samples of used vegetable oil contain high percentages of free fatty acids. In
order to use such sources of oil for biodiesel production, the free acids must first be esterified to
prevent the formation of salts (soap) during the base catalyzed transesterification process.
Methanol solutions of Iron Chloride are known to catalyze the esterification of fatty acids to their methyl esters. However, purification involves reduced pressure distillation which is impractical for large scale biodiesel synthesis. In order to make the iron lewis acid catalyst easier to recover/remove, we are using standard solid phase combinatorial chemistry to link a
phenanthroline molecule to a polystyrene resin system allowing the iron (+3) ion to be available for catalysis, but easily removed by filtration. Early work has focused on the synthesis and purification of the necessary amino phenanthroline which will then be linked to the resin system.
The industrial method for testing the gel point of potential fuels has an elaborate procedure and apparatus. The gel point of a fuel determines whether or not it is usable in an engine. If biodiesel is going to be produced as a potential alternative fuel, it needs to fit the ASTM standards for commercial fuels. An experiment was constructed that attempted to create a simplified method for determining the gel point of biodiesel. A cloud point standard (-6C to -3C) was ordered and our simplified procedure and apparatus were used to determine if the cloud point of the standard could be replicated. Into a test tube 5 mL of the cloud point standard was pipetted and a temperature probe was inserted. The test tube was cooled using a constant temperature bath. Analysis of temperature versus time data produced results that fell within the range of the cloud point standard. This experiment leads us to believe that the gel point of biodiesel can be determined through a simple procedure.
Brittany Krupp, Jenna Howard: Novel method for studying transesterification employing capacitance measurement
A particular interest of biodiesel researchers is a fundamental understanding of factors which influence the kinetics of the transesterification reaction. Such information would allow further investigation into more efficient catalysts and optimization of reaction conditions. The purpose of this study is to evaluate a new method of measuring the kinetics of biodiesel synthesis. Capacitance was measured during the course of the synthesis using copper rods as capacitor plates with the reactants serving as the dielectric. A general trend was found within the first 2-3 minutes of the reaction that is believed to correlate with 90% completion of the reaction. This correlation is in the process of being investigated using GCMS and LC.