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Title: ISOLATION AND CHARACTERIZATION OF T4 BACTERIOPHAGE GP17 TERMINASE: A LARGE SUBUNIT MULTIMER WITH ENHANCED ATPASE ACTIVITY

Author
item Baumann, Richard
item BLACK, LINDSAY - U OF MD MEDICAL SCHOOL

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2002
Publication Date: 2/14/2003
Citation: Baumann, R.G., Black, L.W. 2003. Isolation and characterization of t4 bacteriophage gp17 terminase: a large subunit multimer with enhanced atpase activity. Journal of Biological Chemistry, 278(7):4618-27.

Interpretive Summary: This report describes the purification and characterization of the packaging protein, gp17, from bacteriophage T4. Gp17 has proven difficult to purify because the protein's limited solubility leads to its tendency to form insoluble, inactive protein aggregates, and also it is toxic to host cells that express it. The described procedure results in a preparation that is not only soluble, but very pure and highly active in DNA packaging. Interestingly, we investigated the functionality of gp17 after addition of antiserum raised against a denatured form of the gp17 protein and found the protein preparation was composed of two forms, an active form that could be stimulated in the presence of antibody, and a less-active form. The less active form could be separated by precipitation with the gp17 antibody, which stimulated about four-fold the ATP cleaving activity of the gp17 fraction left behind. After removal of low-activity protein, a significant ATP cleaving activity remained, the highest activity reported for any such protein. The experiments imply significant structural difference between the two forms and also show this protein undergoes a considerable conformational change to form an active complex. These characterizations advance our understanding of the conditions, substrates, and protein- protein interactions involved in activation of the proteins to form the T4 DNA packaging machine.

Technical Abstract: Phage T4 terminase is a two-subunit enzyme that binds to prohead portal protein and cuts and packages concatemeric DNA. To characterize the T4 terminase large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a chitin-binding fusion protein. Following cleavage and release of gp17 from chitin, two additional columns completed purification. The purification yields i) homogeneous, soluble gp17 highly active in in vitro DNA packaging (~10% efficiency, >108 phage/ ml extract); ii) gp17 lacking endonuclease and contaminating protease activities; and iii) a DNA-independent ATPase activity whose Kcat is stimulated >100-fold by the small terminase subunit, gp16 (18 kDa). Analyses also revealed a preparation of highly active and slightly active gp17 forms, and that the latter could be removed by immunoprecipitation using a gp17 antiserum raised against a denatured form of the gp17 protein, leaving gp17 with increased specific activity (~400 ATPs/min/gp17 monomer). This activity approaches that required from the monomer to account for DNA translocation, assuming a multimeric complex consuming 1ATP/2bps DNA packaged. Analysis of gp17 complexes, separated from gp16 on glycerol gradients, showed a prolonged, enhanced ATPase activity persisted after exposure to gp16, suggesting that constant interaction of the two proteins may not be required during packaging.