Advisor |
Biochemistry Research Interests |
|
Dr. Lisa Alex |
Broadly defined, we are interested in how cells transduce environmental changes into metabolic changes; signal transduction. Specifically, we study proteins that participate in 2-component signal transduction (TCST) pathways. In TCST pathways, the first component is an autophosphorylating histidine kinase (HK) whose activity is modulated in response to a stimulus. The phosphoryl group is subsequently transferred to an aspartate residue on a second protein called the response regulator (RR). It's the differential phosphorylation of the response regulator that results in the metabolic change, which could be a modification in enzyme activity, protein-protein interaction, or DNA binding. TCST pathways are found in bacteria, fungi, slime molds, and plants but not in mammals. Because they play a role in virulence in bacteria and fungi they are potential antimicrobial targets. Our group uses the model filamentous fungus Neurospora crassa to study TCST pathways. Among the eleven HKs in N. crassa, we are most interested in understanding the TCST pathway involving the NIK-1. NIK-1, also known as OS-1, was the first HK cloned, sequenced, and knocked out. Dnik-1 mutants display multiple phenotypes such as impaired hyphal development with increased cell lysis and production of carotenoids, decreased female fertility, decreased conidiation, and osmotic sensitivity. They have also been shown by others to be resistant to certain fungicides. We have previously suggested that nik-1 may be involved in regulating proper cell wall development due to the observed Dnik-1 phenotypes. We are using AFM (Atomic Force Microscopy) to investigate the cell wall structures of both wild type and Dnik-1 mutants in various stages of its life cycle (hyphae, conidia, ascospores) under a variety of growth conditions (high salt, low salt, oxidative stress etc.). We are also interested in expressing and localizing NIK-1 in N. crassa. Previous attempts to express this protein by conventional methods in E. coli have failed due to formation of inclusion bodies that could not be refolded. Therefore, we are currently exploring alternative expression systems to obtain purified protein that can be used for phosphorylation and structural studies. In addition, a NIK-1-GFP fusion construct will be used to localize NIK-1 in cells. The predicted protein sequence suggests it does not contain any transmembrane domains, however, it may be membrane associated through interaction with other integral membrane proteins. |
|
Dr. X.C. Sean Liu |
New approach for
regioselective synthesis--proteins as guidance
for regioselective modification of steroids and
disaccharides. The goal of this project is to
establish a new approach of using proteins as
guidance for regioselective synthesis. We will
demonstrate this method by describing two projects
that have strong application potential. One is
to use bovine serum albumin (BSA) to guide the
regioselective reduction of steroids. The other
is to use lectin (concanavalin A) to guide the
regioselective modification of disaccharides such
as lactose and melibiose. Modified steroids, disaccharides
and oligosaccharides play important roles in drug
and vaccine development. This new method may be
useful in the development of new modified steroids
and carbohydrates for developing new drugs and
vaccines. |
|
Dr. Patrick Mobley |
Encapsulated viruses (HIV, influenza, measles...) must fuse their membrane with that of a cell in order to carry out an infection. The viral transmembrane protein mediates the fusion process. Short nonpolar sequences near the amino terminus of viral transmembrane proteins play critical roles in the fusion process and are called fusion peptides (FPs). Our research is directed towards the understanding of how viral fusion peptides cause membranes to fuse. We have used principally the fusion peptide from HIV and the influenza virus. Ongoing projects: Assessing the effect of mutations to FPs on their structure and ability to lyse and aggregate erythrocytes and synthetic phospholipid vesicles. FP structure is studied using FTIR, Circular Dichroism spectroscopy, and computational methods. Fusion and disruption of red cell and vesicle membranes are studied with hemolysis, fluorescence, particle sizing, and dynamic light scattering assays. Screening peptide and nonpeptide inhibitors of FP-induced lysis and aggregation. Some agents can block hemolysis and aggregation of erythrocytes by FP. The HIV fusion inhibitor, T20, is a peptide with a sequence corresponding to part of the HIV transmembrane protein (gp41), and is the best inhibitor we have found to date. T20 inhibits FP-induced hemolysis with an ID50 of 0.5 M. Consequently, we have proposed that T20's inhibition of FP activity is responsible for some part of its efficacy. Determining what other parts of the HIV transmembrane protein (gp41) interact with the membrane. We have found that FP, a sequence immediately preceding the membrane (preTM), and the long N-terminal helix (DP-107) all lyse and aggregate erythrocytes. Other areas to investigate are the intraviral carboxyl terminus and the extraviral loop region of gp41. We would also like to know how these domains of gp41 interact in the fusion process. Defining the amyloid character of FP. Viral fusion peptides have great structural plasticity. They can change from mostly helical to mostly beta sheet with small changes in the environment. This characteristic is shared by the amyloid peptides found in Alzheimer's and Creutzfeld-Jacob diseases. We would like to further explore the similarities between the two families of peptides. |
|
| Dr. Rakesh Mogul | ||
Dr. Peter Oelschlaeger |
Exploring the evolution of metallo-b-lactamases (MBLs) through a combined computational and experimental approach How does the amino acid sequence of a protein determine its structure and function? This is an interesting and challenging question in biochemistry. A related question is how the amino acid sequence of a protein changes over time and improves the protein in terms of stability or activity (evolution). We explore this question for MBLs. These zinc-containing enzymes inactivate b-lactam antibiotics by hydrolyzing them and thus confer antibiotic resistance to pathogenic bacteria. Due to the short life cycle of bacteria, MBLs can evolve rapidly and change their substrate specificity depending on the type of antibiotic they are exposed to (selective pressure). We computationally select MBL variants that could evolve naturally and explore their catalytic efficiency toward different b-lactam antibiotics. So far, molecular dynamics (MD) simulations and computational protein design have been used; in the future we also want to employ reactive force fields. Variants that appear to be better than wild-type enzymes are then characterized experimentally. In the near future, we want to explore NMR and X-ray crystallography (in collaboration with other researchers) to validate some molecular mechanisms that have been proposed for enhanced catalytic efficiency of variant enzymes based on MD simulations. Eventually, including bioinformatics approaches, the goal is to establish a more general scheme for the evolution of MBLs. If we can predict how MBLs evolve under the selective pressure of antibiotics, we may be able to more efficiently plan the application of antibiotics and assist the development of improved antibiotics and MBL inhibitors. Another goal is to transfer this approach to other zinc enzymes involved in disease, such as matrix metalloproteinases, which play roles in certain types of cancer. |
