Scientific Research

General Statement of Research Interest

Throughout my undergraduate education, graduate school and professional experiences, the science laboratory environment was an escape into the wonderful world of inquiry and discovery for me.  Early on, I developed an interest in using biophysical methods to understand how molecules work – particularly macromolecules.  So, during graduate school I concentrated on x-ray crystallography, molecular modeling, spectroscopic methods, hydrodynamic methods, electrophoresis and other enzymological techniques, to study biomolecules – primarily proteins and DNA.

Below, I have presented summaries of selected projects that I have spent considerable amount of time working on and addressing specific scientific questions.  In all my science research studies, I worked in teams with other scientists – sometimes as a leader and other times as a team member. I am now no longer actively involved in science research in the laboratory environment.

Research Projects of Interest

EcoRI-DNA Interactions:  Molecular processes that involve interactions between proteins and nucleic acid sequences, whether specifically or nonspecifically, are central to many biological systems.  I was a doctoral student in a laboratory (at the University of Pittsburgh) that used the EcoRI-DNA system as a model to understand protein-nucleic acid interactions.  This EcoRI endonuclease was chosen as a model for study because:  1. The enzyme (EcoRI endonuclease) is simple and specific, and therefore can be used to cleave DNA into known specific set of fragments.  2. This particular system (EcoRI-DNA) was widely used, and the physical and chemical properties had been extensively studied. 3. The enzyme is composed of small identical subunits (32,000 Daltons), each containing 276 amino acid residues. 4. The 3-dimensional crystal structure of a dodecadeoxynucleotide, CGCGAATTCGCG, containing the cognate recognition sequence (underlined) had been determined and was well-characterized.   

Over the years, several publications have come from these studies and published, shedding light on our understanding of the protein-DNA interactions, as well as the mechanism by which EcoRI endonuclease (a) recognizes its DNA substrate, and (b) cleaves the DNA.

Interferons:  Interferons (IFNs) are cytokines that exhibit a wide range of biological effects including antiviral activity, regulation of cell cycle, anti-proliferative effects, some anti-tumor effects, regulation of production of other cytokines and immuno-regulatory functions.  Their classification is based on cellular origin, chemical properties and antigenicity. The immuno-modulatory functions are, however, specifically associated with interferon-gamma. These interferons are known to exert their effects on cells through specific receptors which are expressed on the surfaces of most cell types. 

There is also a great deal of sequence similarity and topological identity among the gamma-interferon molecules from a variety of animal species – suggesting a common, and perhaps unique, mode of ligand-receptor binding among interferons. I was interested in understanding the molecular mechanism of interferon-receptor interaction/binding, and so I pursued the following specific lines of investigation: 

• Used x-ray crystallography and other techniques to determine the three-dimensional structure of bovine gamma interferon.  I completed this work and published results.
• I also began studies on molecular modeling of interferon-gamma complexed with its receptor.  While these molecular modeling studies were going on, I had an opportunity to start experimental studies on interferon-gamma complexed with its receptor.  To this end, I began crystallization of recombinant rabbit IFN-gamma complexed with a homologue of rabbit IFN-gamma soluble receptor (extracellular domain).   IFN-gamma receptor homologue is a protein secreted by host cells infected by myxoma virus.  This protein behaves very much like IFN-gamma receptor in that it binds IFN-gamma very tightly.  This is a possible mechanism by which a virus has developed ways to counteract interferon production by its host.

C-Reactive Proteins:  A common physiological response to infection, tissue injury or inflammation in most mammals and other species is the systemic production of an acute phase reactant called C–reactive protein (CRP).  Elevated levels of CRP during these acute illnesses and their disappearance as the patients’ conditions improve can be used as an excellent indication of the course of the disease.  My goal was to determine the specific role and mechanism of these CRPs in immune response, infection and tissue injury.  I believed that the determination of 3-dimensional structures was the necessary first step toward this goal.  So, I pursued following experiments:

• Crystallization experiments of dogfish CRP in the presence of both calcium and phosphorylcholine.  Structural information derived from such analyses would provide a clearer picture of the active site.
• Crystallization experiments on rabbit CRP, horseshoe crab CRP and human CRP.  These studies enabled comparisons to be made between mammalian and non-mammalian systems with respect to structure-reactivity relationships among CRPs.
• Analysis of structure to understand oligomerization of CRP monomers in the context of crystal packing.

DNA Methylation:  Sequence-specific DNA methylation in the (DNA) major groove has a marked influence on many genetic mechanisms including (but not limited to) regulation of gene expression, DNA replication, recombination and post-replication proof-reading of DNA, genetic imprinting, X-chromosome inactivation, transposition in Escherichia coli, DNA packaging of some bacterial viruses, host defense against incorporation of foreign DNA in prokaryotes and aging in some mammals.  I was interested in the molecular processes involved in DNA methylation.  Since I had been previously involved in the restriction endonuclease EcoRI-DNA studies, use of the EcoRI methyltransferase which recognizes the same DNA sequence as EcoRI endonuclease would help address some interesting specific protein-DNA recognition questions. 

I initially used molecular modeling to propose the 3-dimensional structure of M.EcoRI based on the known structure of a similar methyltransferase AdoHcy-M.HhaI-DNA complex.  At the same time I proposed the following experiments:

• To determine the crystal structure of M.EcoRI complexed with AdoHcy and DNA oligonucleotides containing the minimum recognition hexanucleotide GAATTC; TGCGCGAATTCGCGC, TCGTGGAATTCCACG and TCGCGAATTCGCG.  Flanking regions are underlined.
• To determine the crystal structure of M.EcoRII complexed with AdoHcy and DNA oligonucleotides containing the minimum  recognition sequence CCWGG (W is A or T);  TGCGCCCWGGGCGC, CAAATTTCC^FWGGATGCGCTC and TCACCCWGGGTG.  Flanking regions are underlined.

Lima Bean Trypsin Inhibitor: There are two primary species of inhibitors present in the soybean seed: the Kunitz type and the Bowman-Birk trypsin inhibitors.  The Bowman-Birk soybean trypsin inhibitor (SBTI) is of considerable interest; this inhibitor has been associated with anticarcinogenic activity when it is consumed in relatively small quantities.  However, SBTI is present in relatively large amounts in the soybean seed.  The high levels of this protein in soybean products result in decreased nutrient availability in the consuming host (humans or domestic animals).  The high thermal stability of SBTI enables most of the inhibitor molecules to survive processing and cooking of soybean products.  Upon ingestion of soy products, large quantities of highly active soybean trypsin inhibitors reach the digestive tract and are able to inhibit the activity of digestive enzymes that are responsible for the breakdown of proteins.  This, in turn, results in a reduced absorption of proteins and amino acids from the digestive tract.

My biomolecular structural studies focused on a Bowman-Birk trypsin inhibitor from lima beans.  I proposed that, based on the observed structural features of LBTI that contribute to its thermal stability, modifications can be made to the related soybean trypsin inhibitor that would result in an altered thermal stability (preferably, a lower thermal stability).  Thus, the target outcome here was to improve the nutritional value of the soybean product by structurally altering an important protein.  My colleagues and I had initial success in some preliminary studies of LBTI using molecular modeling and x-ray crystallography.