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Research Interests
Damaged DNA and its Repair (NIH)
During the last several years, exciting connections have emerged between DNA repair, transcription, and replication. Understanding the repair mechanism has been one of the central subjects in cell biology, since inaccurate repair is the essential step in the production of mutations in a living cell. Without surprise, it has been uncovered that mutations in DNA repair genes cause common forms of hereditary cancer. The precise relationship, however, between the structure and properties of DNA molecules containing each damage and their biological activity or interaction with repair proteins remains to be elucidated.
The structural changes in DNA induced by damage are responsible in part for the harmful effects of these lesions and at the same time present recognition sites for repair enzymes. Therefore, the efficiency of repair is likely to depend on the extent to which these changes alter the structure of the DNA, making it recognizable for the repair enzymes involved. This depends, in part, on the extent to which the individual damage affects the template structure, also on the DNA sequence context and on the efficiency of the polymerase and repair enzymes involved.
The overall goal of the proposal is to understand at the molecular level the specific DNA lesions: UV damage, thymine glycol and 8-oxo guanine. We will then investigate the three damage related enzymes, XPA/RPA, Pol h, Pol i and rpS3, to understand their interaction with the specific lesions. The atomic coordinates of lesion-containing DNA and the DNA/enzyme complex provide great starting material for the investigation of recognition and/or reaction mechanisms in the cell.
Calsequestrin, Its Implication for Calcium Regulation and Its Interaction with a Membrane Protein, Junctin, Triadin and Ryanodine Receptor (NSF)
Ca2+ regulation is coupled to critically important cellular processes in such complex cellular functions as contraction, secretion, fertilization, proliferation, metabolism, heartbeat and memory. Central to these functions is the ability of Ca2+ to act as a ubiquitous, powerful trigger of protein conformational change. In this process of Ca2+ regulation, calsequestrin (CSQ), which has more than 45 Ca2+ binding sites, and similar proteins found in the endoplasmic reticulum and sarcoplasmic reticulum, serve as Ca2+ storage proteins.
Many physico-chemical studies have been conducted to elucidate structure/function relationships regarding calcium regulation by these highly acidic (40% Asp+Glu) proteins. These studies were limited by a lack of detailed structural information until we determined the first 3D crystal structure of CSQ. The observed structure is ripe with implications for the detailed mechanism of CSQ function and the structural changes that underlie calcium regulation.
According to our new, crystal structure-based structure/function model, as the Ca2+ levels increase and monovalent ion (H, K, Na) levels decrease in the SR lumen, there is a concomitant formation of CSQ polymers or aggregates and high capacity Ca2+ binding. The focus of study is to directly test the molecular details of our model using both physical chemistry methods and recombinant genetic techniques. Based on our unique crystal structures and preliminary data, we are investigating the significance of CSQ folding and polymerization with respect to physiological function and the role of structural domains within the CSQ and other related proteins.
Structural Studies of phenylpropanoid Biosynthetic Process (NIH)
Lignins, lignans and isoflavonoids are major constituents of vascular plants and account for nearly 30% of the organic carbon circulating in the biosphere. They play important roles in plant defense, helping hosts discriminate between biological pathogens and symbiotic non-pathogens. This research project focuses on determining the structures of various key proteins and enzymes involved in lignan and isoflavonoid biosynthesis, namely the so-called dirigent protein and three reductases: pinoresinol-lariciresinol reductase, phenylcoumaran benzylic ether reductase and isoflavonoid reductase.
In addition to their physiological roles in plants, these compounds have pronounced antimicrobial, antifungal, antiviral, antioxidant and anticancer properties. Certain isoflavonoids and lignans are used in medicinal applications, especially for the clinical treatment of various cancers and in cancer prevention. It is generally accepted that low cancer incidence rates in people with specific plant-based diets is due to the so-called chemopreventive effects of particular lignans and isoflavonoids. For these reasons, there is considerable interest in making these compounds more generally available by defining and exploiting the lignan and isoflavonoid biosynthetic pathways. These approaches are seriously hampered, however, by a lack of detailed information on the enzymes involved and the limited success in obtaining them in significant amount. The goal of this project is thus to define the structure-function relationships of these key proteins and enzymes in lignan and isoflavonoid biosynthesis through high-resolution X-ray structural analyses. The information gained from these studies will be very useful for exploring the regulation of lignan and isoflavonoid biosynthesis for ecological plant protection, and for the industrial-scale regiospecific and stereospecific synthesis of these pharmacologically active substances.
Protein Thermostability and Redox Chemistry :
- Thermostability: Compared to their mesophilic counterparts, thermophilic enzymes have a few kcal/mol additional stabilization energy. The origins of this thermostability should lie within interactions between the constituent amino acid residues, plus cofactor and solvent. In principle, a thorough analysis of the numbers and types of interactions between these constituents may suggest the elements contributing to stabilization and the knowledges learned through such experiments could be used directly in the design of thermostable enzymes. Unfortunately, despite the recent isolation and structural studies of several thermophilic/mesophilic protein pairs, the general structural features or the molecular determinants of protein thermostability that account for the higher thermal stability of thermophilic proteins has not been fully identified.
Here we are investigating structural features that could lead to stabilization of the thermophilic over the mesophilic enzymes through a systematic approach. To study the factors leading to stabilization, we are comparing the detailed structure and thermostability of a number of systematically chosen proteins from a mesophilic and a thermophilic organism.
- Redox Chemistry: Electron transfer reactions are the most essential and the simplest processes for life. Not only do they provide the means for transforming solar energy and chemical energy into an utilizable form for all living organisms, they also extend into a range of metabolic processes that support the life of the cell. In life, redox proteins are necessary to control these rates. Differences in "E" of up to a few hundred mV are seen between homologous redox proteins with the same redox site. The site-directed mutagenesis of a single residue can drastically modify this electric potential and subsequently the electron transfer rate. The basic questions, then, are: what are the physical factors that determine the rate constants for protein electron transfer? How do protein structure, and the structure of the redox center itself, influence these factors? To understand this, electron transfer proteins must be studied at an atomic level. We have made systematic mutations at various sites in rubredoxin and ferridoxin to see how these changes influence the redox potential and the local structures. The systematic studies of the crystal structures and the measurement of redox potential could form an essential bridge for electron transfer proteins between their general and the particular behaviors.
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