My main research interests lie in understanding the molecular basis of protein function. Of particular interests are studies of metalloenzymes structure/function, protein electron transfer, protein-protein interactions, and receptor-ligand interactions that are involved in biological signal transduction.
My current projects aim to understand the mechanism of nitric oxide (NO) synthesis by human and plant nitric oxide synthases (Nos) and reactive oxygen species (ROS) production by non-phagocyte NADPH oxidase enzymes (Nox). Nos and Nox play important roles in the control of fundamental physiologic processes, including vascular tone, platelet function, neuron signaling, mitosis, apotosis, and cell migration. Understanding how auxiliary cellular components and factors interact with enzymes to control their biochemical reactions presents exciting challenges.
Research interests include:
Biochemistry of non-phagocyte Nox5 and Duox
Nox proteins share similar sequence features, consisting of a polypeptide homologous to gp91phox that contains a five-to-six transmembrane heme domain and a flavoprotein or dehydrogenase (DH) containing FAD and NADPH binding sites. An active complex formation from accessory and/or regulatory proteins are required for Nox1-3 while Nox4 is constitutively expressed as an activated form. Interestingly, superoxide and hydrogen peroxide production by Nox5 and Duox are Ca2+ dependent, through their additional self-contained N-terminal EF-hand domain (EFD). Nox5 overexpression results in high ROS production and has been reported to be associated with disease development, including hairy cell leukemia, prostate cancer, diabetic nephropathy, vascular diseases.
Based on our characterization, Nox5's EFD functions simialr to CaM. The following are the models of their holo forms (left: Nox5's EFD; right: CaM) based on homology modeling. Red: negative charges Blue: positive charges Gray: neutral charges
Structural and functional studies of plant NO synthase
Tetrahydrobiopterin roles in human Nitric Oxide Synthases
Calmodulin in STIM Ca2+ Storage and Fas Receptor
Lysozyme and Chitin related projects
Techs and Instruments used in our research
Recombinant Protein Expression/Purification
The proteins characterized in our lab are produced by recombinant tech and purified with chromatography. The following is an example of CFP-fused protein that is purified with FPLC
Isothermal Titration Calorimetry (ITC)
Our lab equips with a MicroCal VP-ITC calorimetry, which is used to obtain thermodyanic parameters of binding.
The following is a recoding of ITC experiment during the titration of ligand to receptor solution. In the begining, the system takes time to reach temperature equilibirum followed by several injections. This is an endothermal reaction.
Time-Correlated Single Photon Counting (TCSPC)
We use TCSPC for fluoresence lifetime measurement (~ nsec) for specific probe(s), which allows us to deduct the emitted components and determine the energy transfer yield from FRET.
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Last update: 09/10/2010
