This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Phages are classified mainly by their morphology, but an evolutionary relationship of all tailed phages is well known. The virions of Bacteriophage Sf6, HK620, HS and APSE-2 resemble to those of phage P22 with an exception of one structural protein. The length of the cell penetrating tail needle gp26 significantly varies among homologs in these virions. The tail needle gp26 in phages is functionally equivalent to the well characterized tail needle of phage P22, mediating viral DNA injection in to host cells. It is plausible, despite having same functional role; modes of penetration and DNA injection through lipid bilayer could be substantially different across homologs. Using CHESS facility, we have recently solved a 2.1A X-ray structure of P22 gp26, providing structural basis for penetration and DNA injection. To enrich our understanding about phage DNA injection mechanisms we have done a recombinant expression of all gp26 homologs from Sf6, HK620, HS and APSE-2 in E. coli as a soluble protein. All gp26 homologs from various phages were purified to homogeneity. A preliminary characterization has been carried out using various biophysical techniques. For structural studies gp26 homologs were concentrated to 4-7 mg/ml and used to set up Sigma-Aldrich Crystal Screen using hanging drop vapour diffussion method. For HK620 gp26, the best crystals were obtained at pH 9.0 using TAPS as crystallization buffer and either 12-16% PEG 8000 or 16-26% PEG 4000 in the presence of 0.1 M ammonium sulfate. In both cases gp26 crystals grew within two week at 293 K to reach maximum dimensions of 50 x 100 x 500 m. Using similar screen, we got positive hits for SF6 and HS gp26 as well, and we are in the process of optimizing conditions by varying PEGs to achieve morphologically better looking crystals.
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