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Mechanism of Stimulation of the DNA-packaging ATPase in Bacteriophage T4

Mechanism of Stimulation of the DNA-packaging ATPase in Bacteriophage T4. Karoly Viragh Comprehensive Seminar March 13, 2003. Overview. Introduction Bacteriophage T4 DNA packaging Arginine-finger hypothesis Experimental Design Peptide design and synthesis

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Mechanism of Stimulation of the DNA-packaging ATPase in Bacteriophage T4

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  1. Mechanism of Stimulation of the DNA-packaging ATPase in Bacteriophage T4 Karoly Viragh Comprehensive Seminar March 13, 2003

  2. Overview • Introduction • Bacteriophage T4 DNA packaging • Arginine-finger hypothesis • Experimental Design • Peptide design and synthesis • Preliminary evaluation of ATPase activity • Results and Discussion • PCR-amplification and purification of coding DNA • Cloning • Peptide Expression and Purification • ATPase assay • Conclusions • Questions

  3. Intro – Bacteriophage T4 Viruses - obligate parasites, which depend on the host cells that they infect to reproduce Bacteriophages - viruses that infect bacteria T4 - specifically infects E. coli Negative Contrast Electron Micrograph of T4

  4. Head Assembly of Bacteriophage T4

  5. Intro – T4 DNA packaging • T4 DNA packaging • The process of moving the 170 kbp genomic dsDNA into the empty capsid and its subsequent organization • T4 Packaging Machine: • gp20 (61 kDa structural component – portal vertex) • gp17 (70 kDa large terminase/packaging protein) • gp16 (18 kDa small terminase/packaging protein)

  6. Intro – GTPase catalytic center Arginine finger(s) in Ras and G-proteins Recent analyses of G-protein GTPases suggest that the catalytic stimulation of GTP-hydrolysis is due to a precise positioning of one or more arginine residues (“arginine fingers”) of a GAP (GTPase Activating Protein) into the GTPase catalytic center

  7. Sequence analysis of the gp16 subunit of four bacteriophages in the T4 family identified strictly conserved arginine residues: R41, R53 and R81 T4: M-----EGLDINKLLDISDLPG-IDGEEIKV-YEPLQLVEVKSNPQNRTPDLEDDYGVVRRNMHFQQQMLMDAAK 68 RB49: M--------KQLMNFESLGLPGSFDAEEDRVSYDPLVLTPVESHPEDRNIDLQRDYNEARQNIHFQNQMMMDAAK 67 KVP40: MNDELMQQLQALTQVDGLDLPGAIEAPEPEE-FQPPVIKEVESHPTERVKDLEADYATVRDNAHFQQQLLRMAAL 74 KVP20: MNDELMKQLQALTQVDELDLPGAIEAEEPEE-FQPPVIKEVESHPSERVKDLESDYATVRDNAHFQQQLLRMAAM 74 Con1: M--------------+---LPG--+--E----+-P--+--V+S+P--R--DL+-DY---R-N-HFQ-Q++--AA- T4:IFLETAKNADSPRHMEVFATLMGQMTTTNREILKLHKDMKDITSEQ-VG--TKGAVPTGQMNIQNATVFMGSPTE 140 RB49:IYLELAKNSESPRFLQAFNGLMQQMSNNNKELLNIHKDMKKIT-EQ-AGEKKKDNTPAAPVNIQNATVFMGSPSD 139 KVP40:KAFENASMSDAPRMMEVFATLMTQMTNNNKQILDIQKQMKDITQQEIASPQGGGGGTVQSINAETA-VFVGNARD 148 KVP20:KAFENASMSDAPRMMEVFATLMTQMTNNNKQILDIQKQMKDITQQEIATAQGGSGGTVQSINAETA-VFVGNSRD 148 Con1: ---E-A--+++PR-++-F--LM-QM+--N+++L-+-K-MK-IT-++--------------+N-+-A-VF+G+--+ T4: LMDEIGD-----------AYEAQEAREKVINGTTD 164 RB49: LMDEIED---------------EEAR--VIEGETV 158 KVP40: LLNEVGSRQEYLRNKKEEEIIDVEPEEKVQEKDD- 182 KVP20: LLNEVGSRQEYLRSKREEEIIDVEPEEKVQEKDD- 182 Con1: L++E+------------------E----V------

  8. Intro – Hypothesis • Formation of the gp16-gp17 enzyme complex • allows the positioning of one or more arginine fingers from gp16 into the gp17 ATPase catalytic center, which • stimulates ATPase and DNA packaging activities by stabilizing the transition state for ATP hydrolysis

  9. ExperimentalDesign • Site-directed mutagenesis (not used) • Peptide-modeling approach (employed here) • Design truncated gp16-peptides based on 2° structure predictions by a variety of online computational tools • Synthesize peptides using a molecular genetic approach (Peptide 2) • PCR-amplify the gene coding for Peptide 2 • Purify the amplified DNA fragment • Clone the DNA fragment into a plasmid vector • Induce the expression of Peptide 2 from the vector • Purify Peptide 2 using Ni2+-Agarose chromatography • Evaluate ATPase activity (preliminary results)

  10. Results – PCR Amplification PCR amplification of the peptide-coding regions from T4 genomic DNA (P1=Peptide 1, P2=Peptide 2)

  11. Results – DNA purification Agarose gel purification of the DNA fragment coding for Peptide 2 (P2)

  12. Results – Cloning Testing for the presence of the right insert in the correct orientation by PCR

  13. Results – Induction SDS-PAGE analysis of IPTG-induced protein expression

  14. Results – Peptide Purification SDS-PAGE analysis of the purification process for Peptide 2

  15. Results – ATPase assay ATPase assay to evaluate the activity of the truncated gp16 protein (Peptide 2)

  16. Conclusions • T4 DNA packaging is a complex process involving the small terminase subunit gp16. • The critical segments of gp16 can be evaluated using the peptide modeling approach. • Peptide 2 does not stimulate gp17 ATPase activity. • More complete biochemical analysis is needed.

  17. Acknowledgements • I’d like to thank: • Dr. Rao, for his invaluable tutelage throughout the experiments; • Dr. Kovach, for her assistance in revising the report; • Dr. Kondabagil, for his help in protein purification and ATPase assays

  18. On a final note… This presentation and the comprehensive paper are available on-line at http://www.karoly.tk

  19. Any questions?  Thank you for your time!

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