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CURRENT & RECENT TRAINEES

Lawrence Borketey (Schnarr Research Group)

First Research Project
Polyketides are pharmacologically active, natural products biosynthesized by some soil bacteria using large, multI modular enzymes called polyketide synthase (PKS). A module of a PKS is made up of ketosynthase (KS), acyltransferase (AT), acyl carrier protein (ACP) - all of which are responsible for a single round of two carbon chain elongation- ketoreductase (KR), dehydratase (DH) and enoylreductase (ER). KR, ER and DH are responsible for functionalizing the growing polyketide chain. KR converts the β-carbonyl group on the polyketide chain to a hydroxy group, DH dehydrates the hydroxy product of KR activity into an olefin whiles ER reduces the olefinic DH product to a saturated group. By controlling the activity of these functionalization domains (i.e. KR, DH, ER), it is possible to produce a wide variety of polyketides distinct from those produced by the natural bacteria and hence increase the repertoire of compounds available for rational drug design.

As a proof of principle, a simple PKS module comprising all six domains is being used to biosynthesize four different δ-lactones. In one enzyme, all six domains are present and active. In the other three mutants, one of KR, DH or ER has been deactivated via active site mutagenesis. This simple PKS module utilizes 3-hydroxy-valeryl coenzyme-A as starter unit, malonyl coenzyme-A as extender unit and NADPH as cofactor for both KR and ER. The N-acetyl cysteamine thioester of 3-hydroxy valeric acid is used as an analog of 3-hydroxy valeryl coenzyme-A. This thioester was synthesized from methyl-3-hydroxy valerate by first hydrolyzing the ester to the acid followed by coupling to N-acetyl cysteamine. An assay has been developed for the wild type enzyme and 4-ethyl-δ-lactone was detected by GC/MS as the product. Currently, I am working on the biosynthesis of the other δ-lactones using the mutant enzymes.

Second Research Project
6-deoxyerythronolide B (6-dEB), the precursor of the antibiotic erythromycin, is biosynthesized by the enzyme 6-deoxyerythronolide B synthase (DEBS). DEBS requires a single starter unit (propionyl coenzyme A) and six extender units (methyl malonyl coenzyme) for the total biosynthesis of 6-dEB. The extender unit is thus amplified in the final 6-dEB product. Both the starter and extender units are also produced by the action of propionyl coenzyme A carboxylase (PCC) on propionic acid. It is therefore possible to place a tag on the propionic acid and this tag will be amplified in the final 6-dEB product.

To this end the propionic acid is tagged with fluorine. (The fluoropropionic acid was synthesized by the Jones oxidation of 3-flouropropanol) The 6-dEB product will thus be heavily fluorinated. The fluorine tag provides a
facile means for the isolation of the fluoro-6-dEB via fluorous extraction. Successful tagging of 6-dEB will be useful in the detection of methyl malonate derived metabolites in any bacteria. Fermentation of the bacteria in question with fluorinated propionic acid (in a bacteria which utilizes PCC) or fluoro methyl malonyl coenzyme-A (in a bacteria which lacks PCC) will produce fluorinated metabolites which can be isolated using the fluorous extraction technique.

I have been able to optimize the fermentation condition required for 6-dEB production by an engineered E. coli strain transformed with DEBS. These same conditions have also been used to produce the flouro-6-dEB. 6-dEB has been characterized by LC-MS. I am now working on developing a satisfactory method using the same technique to characterize my flouro-6-dEB.
 
Mariel Feliciano (Chambers Research Group)
The study of neuronal receptors using photochemistry, electrophysiology and other organic and biomolecular echniques
 
David Griffin (Bhatia Research Group)

Self-assembling polymer hydrogels have numerous advantages that make them ideal candidates for use as biomaterials. High water content, desirable pore structures and bioresorbability are just a few of the characteristics that these systems share with native human tissues. Tuning the structure of hydrogel polymers or adding a second component to create nanocrystalline domains forms composites similar in structure to naturally occurring tissues and materials.

My research focuses primarily on the synthesis and characterization of biomimetic nanocomposite materials. Additionally, I have conducted mammalian cell culture analysis to determine the effectiveness and cytotoxicity of selected biomaterials. These studies were carried out using the HepG2 (human hepatocellular liver carcinoma) cell line and primary bovine chondrocytes that we isolated from calf knee joints.

Materials that I am researching are similar in composition, morphology and physical properties to bone, one of the most ubiquitous examples in nature of biocomposites and biomineralization. For my investigation I am determining how environmental factors influence the in situ formation of calcium phosphate crystals in block copolymer gels. This parallels the growth of bone, which is formed by ion diffusion and apatite mineral growth on oriented Type I collagen polymers.

Factors such as pH, temperature and ionic strength influence the mineralization process in many ways. These effects range from determining the type and size of crystals produced to the overall mineral content of the composite. Because environmental and experimental variables determine the final composition and structure and thusly the physical properties of the material, a great deal of work is focused on characterizing the composites after they are formed.

In order to determine what calcium phosphate crystal phases are present in my samples I conduct powder X-ray diffraction (XRD) on the composite mineral. Energy dispersive X-ray spectroscopy (EDS) discerns relative ion (i.e., calcium to phosphate) ratios and can substantiate the results of XRD. Scanning electron microscopy (SEM) is used to investigate the overall micro- to nanostructure and particle size distribution of the mineral particles. Bulk mechanical properties are tested rheologically to illustrate the effect of calcium phosphate crystals on the elastic modulus of the gel.

By applying fundamental principles of chemistry to tune in situ nanocomposite formation we are able to produce biomaterials that may have improved biological functionality. Establishing the biocompatibility and effectiveness of these composites through cell culture experimentation techniques and biological assays will give us the final word on the direction we will take to create more useful biomaterials.
 
Seena Koshy (Thompson Research Group)
The molecular mechanism of transmembrane signaling in bacterial chemotaxis system as mediated by membrane proteins
 
Fabian Romano (Heuck Research Group)

The problem of protein translocation across biological membranes has remained in the battlefront of science for the last 35 years. Although remarkable progress has been achieved, salient questions remain to be answered. Protein translocation through mitochondrial, chloroplasts and Gram-negative bacterial membranes share all similar mechanisms that have been widely studied. However, a question of public health relevance that is only partially answered is the following:  How proteins synthesized in the cytoplasm of pathogenic bacteria are able to reach, in an active way, the cytoplasm of their eukaryotic host cells? In Gram-negative bacteria, this translocation process is carried out through the Type 3 Secretion System (T3SS), a highly conserved secretion system composed by more than twenty different proteins. The T3SS resembles a nano-syringe able to engage the host plasma membrane and allow: 1) The secretion of toxic proteins through both bacterial membranes, and 2) The translocation of the bacterial toxins through the host plasma membrane at the “syringe” tip/membrane interface. In P. aeruginosa, PopD and PopB, two bacterially encoded proteins are thought to permeabilize the host plasma membrane to allow translocation through the T3SS. This pair of proteins is highly conserved among Gram-negative bacteria, suggesting a common mechanism for protein/toxin translocation. Deletion of either of them completely abolishes bacterial pathogenicity. Also, it is known that each protein alone is able to form pores in liposomal membranes. Because of their key role in protein translocation through the T3SS, these proteins are currently under characterization in Dr Heuck’s laboratory, were I work towards my PhD degree.

In order to understand the overall process of protein translocation, it is required to study how PopD is able to spontaneously bind to membranes and to form pores. Additionally, it is important to study whether PopD conformation in the membrane is affected by other proteins involved in the translocation process (i.e. PopB).  For this reason, the focus of my research is to investigate the topology of membrane bound PopD, and how this topology is affected by other protein factors involved. My goal is to understand how PopD is able to bind to membranes and permeabilize them to allow protein translocation.

To obtain this type of information, I currently use an in vitro biochemical/biophysical approach. Briefly, I use full-length wild type proteins and various functional single-cysteine mutants of PopD at the segment to be studied. Then, I attach a low molecular weight fluorescent dye to PopD single cysteines, and obtain structural data by combining different fluorescent techniques such as fluorescence quenching and fluorescence lifetime. These experiments are carried out on liposomal membranes that resemble biological membranes in their lipid composition.

Using this approach, we are starting to obtain valuable topological data of membrane bound PopD.