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

2. 1.Introduction Bacteriophage T4 DNA packagingBacteriophage T4 DNA packaging Arginine-finger hypothesisArginine-finger hypothesis 2.Experimental Design Peptide design and synthesisPeptide design and synthesis Preliminary evaluation of ATPase activityPreliminary evaluation of ATPase activity 3.Results and Discussion PCR-amplification and purification of coding DNAPCR-amplification and purification of coding DNA CloningCloning Peptide Expression and PurificationPeptide Expression and Purification ATPase assayATPase assay 4.Conclusions 5.Questions Overview

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. 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) Intro – T4 DNA packaging

6. 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 Intro – GTPase catalytic center

7. 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------ Sequence analysis of the gp16 subunit of four bacteriophages in the T4 family identified strictly conserved arginine residues: R41, R53 and R81

8. Intro – Hypothesis Formation of the gp16-gp17 enzyme complex 1.allows the positioning of one or more arginine fingers from gp16 into the gp17 ATPase catalytic center, which 2.stimulates ATPase and DNA packaging activities by stabilizing the transition state for ATP hydrolysis

9. Experimental Design A.Site-directed mutagenesis (not used) B.Peptide-modeling approach (employed here) 1.Design truncated gp16-peptides based on 2° structure predictions by a variety of online computational tools 2.Synthesize peptides using a molecular genetic approach (Peptide 2) a.PCR-amplify the gene coding for Peptide 2 b.Purify the amplified DNA fragment c.Clone the DNA fragment into a plasmid vector d.Induce the expression of Peptide 2 from the vector e.Purify Peptide 2 using Ni 2+ -Agarose chromatography 3.Evaluate ATPase activity (preliminary results)

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

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

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

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

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

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

17. Conclusions 1.T4 DNA packaging is a complex process involving the small terminase subunit gp16. 2.The critical segments of gp16 can be evaluated using the peptide modeling approach. 3.Peptide 2 does not stimulate gp17 ATPase activity. 4.More complete biochemical analysis is needed.

18. 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

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

20. Any questions? Thank you for your time!