The Class Of Sulfur Redox Protein Three-dimensional Crystal Structure Of The Catalytic Domain And Its Functional Study

Human thioredoxin-like gene (hTRXL), which shows variation in expression level related to the development of human fetal brain was isolated independently in our lab. The gene has been expressed both in E. coli and in eukaryotes. Moreover, northern blot and insulin disulfide reduction assay have been carried out to identify the gene products. In this dissertation, an attempt is made to explore the relationships between the three-dimensional structure and biological function by researching on the crystal structure of hTRXL.According to the experimental requirement of crystallography, hTRXL full-length gene as well as the fragment of hTRXL N terminal domain (residues 1-105) was transferred from pET28b vector, which had been used to create histidine-tagged fusion proteins for insulin disulfide reduction assay, to pEGX-4T-3 vector. GST fusion proteins were over expressed in E. coli strain BL21. Glutathione S-transferase (GST) Gene Fusion system was use to obtain the target protein with purity higher than 90%.Crystal of hTRXL-N in monoclinic crystal form (C2) was successfully grown using the method of hanging-drop vapor-diffusion. Native data were collected on MAR345 to 2.22 A. The crystal structure was solved by molecular replacement and difference Fourier analysis in X-ray crystallography, and then, was refined smoothly alternating steps of automatic and manual adjustment. The final model has reasonable crystallographic R and R_free values with good stereochemistry and the mainchains fit well with the electron density map.Despite the amino acid sequence of hTRXL-N shows comparatively low identity to that of thioredoxin from different species, the three-dimensional structure is similar. Moreover, the possible interaction surface for substrates in hTRXL-N is very similar to the corresponding region in hTRX-Ref-1 complexes. All the data from the crystal structure further confirm the previous results of the functional analysis that we have got...