Crystallographic and Cryo-EM Studies of CHL1, SMN and Thioesterase I
Nitrate transporter CHL1 from Arabidopsis is unique in that it was found to possess both high and low affinities to nitrate. CHL1 is a membrane protein which utilizes free energy stored in proton gradient to drive the transport of nitrate into cells. Sequence comparison has shown that this protein is similar to a peptide transporter family. However, CHL1 does not transport the peptides tested. In this project, we attempt to over-express and crystallize the CHL1 protein. Our investigation will assist in understanding the molecular mechanism of transporters in this family.
Spinal muscular atrophy (SMA) is a leading fatal autosomal recessive disease. Positional cloning has identified that survival motor neuron gene (SMN) is responsible for this disease. The SMN showed no sequence identity with previously identified genes. It is rather surprising to find that this protein is located in a subnuclear structure called gem (gemini of coiled bodies), implicating its function is related to RNA metabolism. Further investigation also shows this protein can associate with hnRNP or spliceosomal snRNAs, which are involved in RNA processing. The interaction of SMN with anti-apoptotic factor Bcl 2 also suggests SMN may be involved in programmed cell death. We want to crystallize this protein and determine its structure. Information gained in this study will help us to understand how the SMN protein forms oligomers and how it interacts with other proteins and RNA. The structural information may also assist in finding a way to cure spinal muscular atrophy.
Recent advances in molecular biology and cryo-electron microscopy have opened up opportunities for studying a wide range of interesting and complex biological systems. Especially, many biological systems have large protein complexes formed by a large number of proteins to exert their biological function. Structural study of single component is not sufficient to reveal the structure-function relationship of a complete system.
We aim to build a state-of-the-art modern cryo-electron microscopy research facility at Academia Sinica. Proposed equipment purchases including a 200kV FEG electron microscopy, a 4k x 4k CCD, a vitrobot, etc. The EM room will be renovated to reduce acoustic noise, vibration, barometric pressure change, room and chilled-water temperature variations, and magnetic field variations. Initial research projects are proposed: (1) studies of 3D structure of Hepatitis Delta Virus; (2) studies of E. Coli exosome and degradosome and its complexes; (3) other on-going research projects in Academia Sinica.