Sandbox Reserved 598: Difference between revisions

In determining the three-dimensional structure of the Jak2 protein, three primary methods of analysis were used; protein expression and purification, crystallization, and x-ray data collection. <ref> Lucet, I., Fantino, E., & Styles, M. (2005). The structural basis of janus kinase 2 inhibition by a potent and specific pan-janus kinase inhibitor. Blood, 107, 176-183. doi: 10.1182/blood-2005-06-2413 http://bloodjournal.hematologylibrary.org/content/107/1/176.full.pdf </ref> In the protein expression and purification the Jak2 residue was cloned using pFastBac which uses “two promoters in a single vector for expression of two proteins simultaneously in insect cells”. <ref> http://www.invitrogen.com/1/1/14896-pfastbac-dual.html </ref> The bacmid DNA with the kinase insert was then isolated and put into cells of insect army worms. The cells were then grown, lysed and centrifuged after which the protein was then incubated, separated with gel filtration and fractions were taken for crystallization trials. In these crystallization trials, the protein residue was used to grow crystals via hanging drop vapor-diffusion. The purified protein complex was then mixed with solutions which subsequently formed crystals after one to three days. The crystalized protein was then flash frozen and the structure  determined via molecular replacement and the AmoRe program. <ref> ) Lucet, I., Fantino, E., & Styles, M. (2005). The structural basis of janus kinase 2 inhibition by a potent and specific pan-janus kinase inhibitor. Blood, 107, 176-183. doi: 10.1182/blood-2005-06-2413 http://bloodjournal.hematologylibrary.org/content/107/1/176.full.pdf </ref>

[[Image:Jak2structure.png|thumb|300px|left|Ribbon representation of Jak2 structure as determined by Dr. Isabelle S. Lucet et al]]

The structure of Jak 2 can be broken down into seven separate components, as seen in the pictured diagram to the left. The top, gray, portion of the protein was found to be the N-terminal lobe (residues 840-931). This loop is comprised of a 5-stranded anti-parallel beta-sheet (Beta1 - 5) and one alpha-helix (alpha C). The large dark green portion seen on the bottom is the COOH-terminal lobe. This carboxylic acid lobe is comprised of 8 alpha-helices (alpha D-alpha K), and 3 3/10 helices (3/10B, 3/10C, 3/10D), and 3 pairs of anti-parallel Beta-strands (Beta7-8, 6-9, and 10-11). The orange portion in the middle-right of the protein is the glycine loop which makes contacts with the activation loop and catalytic loop. This glycine loop, while small, is of great importance, as it is known to be essential in substrate and nucleotide binding. In yellow, to the top-left, there is a hinge region present which aids in molecule interactions. The blue section in the low middle is the catalytic loop, the red loop to the right is the activation loop and finally the dark blue section towards the bottom-right is the JAK2 lip which contains one 3/10C helix and one alpha-helix connected by a short linker. <ref> ) Lucet, I., Fantino, E., & Styles, M. (2005). The structural basis of janus kinase 2 inhibition by a potent and specific pan-janus kinase inhibitor. Blood, 107, 176-183. doi: 10.1182/blood-2005-06-2413 http://bloodjournal.hematologylibrary.org/content/107/1/176.full.pdf </ref>