Mechanism of Stimulation of the
DNA-packaging ATPase in Bacteriophage T4
Karoly Viragh
Comprehensive Seminar
March 13, 2003
Overview
1.
Introduction
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•
2.
Experimental Design
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•
3.
Peptide design and synthesis
Preliminary evaluation of ATPase activity
Results and Discussion
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•
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4.
5.
Bacteriophage T4 DNA packaging
Arginine-finger hypothesis
PCR-amplification and purification of coding DNA
Cloning
Peptide Expression and Purification
ATPase assay
Conclusions
Questions
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
Head Assembly of Bacteriophage T4
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)
Intro – GTPase catalytic center
Arginine finger(s) in
Ras and G-proteins
Recent analyses of G-protein
GTPases suggest that the
catalytic stimulation of GTPhydrolysis 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
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------
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
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)
3.
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 Ni2+-Agarose chromatography
Evaluate ATPase activity (preliminary results)
The predicted secondary structures for gp16, as well as the two peptides: Peptide 1 (68
amino acids from 27 to 94) and Peptide 2 (73 amino acids from 47 to 119) [H=alpha-helix,
E=extended(beta)-sheet, –= random coil, T=beta-turn, L=omega-loop].
0.
1.
2.
3.
4.
5.
6.
1
2
3
4
5
6
MEGLDINKLLDISDLPGIDGEEIKVYEPLQLVEVKSNPQNRTPDLEDDYGVVRRNMHFQQ
------------------------------E-------------HHHHHHHHHHHHHHHH
-----H----------------E------HHEE-----------------HH---HHHHH
LLL---L-----LLLLLLLL-EEEEE---EEEEE-LLLLLLLL-LLLL---------HHH
HTT----HEE-----TT-----EEEE--HEEEEE-------------HHHEEHHHHHHHH
---------------------EE------EEEEE---------------HHHHHHHHHHH
-----HHHHH-----------EEEE----EEEEE-------------HHHHHHH--HHHH
---HHHHHHHH---------HHHHHHHHHHHHHHH-T-------HH----EEHHHHHHHH
0.
1.
2.
3.
4.
5.
6.
7
8
9
0
1
2
QMLMDAAKIFLETAKNADSPRHMEVFATLMGQMTTTNREILKLHKDMKDITSEQVGTKGA
HHHHHHHHHHHHH-------HHHHHHHHHHHHHHH-HHHHHHHH----HHHHHHH----HHHHHHHHHHHHHH--------HHHHHHHH-------HHHHHHH---------E-----HHHHHHHHHHHHHH---LL-HHHHHHH-HH------HHHHHHHH---------E-----HHHHHHHHHHHHH--------HHHHHHHHHHHH---HHHHHHHHHHHHHHHHH-ETTT-HHHHHHHHHHHHHH-------HHHHHHHHHHHHHHHHHHHHHHHH----HHHHH-----HHHHHHHHHHHHH-------HHHHHHHHHH----HHHHHHHHHHHHHHHH---------sHHHHHHHHHHHHHHHHH---HHHHHHHHHHHHHH--HHHHHHHHHHHHHH-HHHHT----
0.
1.
2.
3.
4.
5.
6.
3
4
5
6
VPTGQMNIQNATVFMGSPTELMDEIGDAYEAQEAREKVINGTTD (164 amino acids)
-----EEE--EEEEE----HHHHHHHHHHHHH---EEEE---------EE----EEE-------HHHH---HHHHHHHHHEE------L---E----EEE-LL-HHHHHHHHHHHHHHHHHHHHH-LLLL
--------TT-EEEE---HHHHHHHHHHHHHHHHHHHHHHT-----------EEEEE-----HHHHHHHHHHHHHHHHHHHEEEEE-----EEE---EEE---HHHHHHHHHHHHHHHHHHHHHH--------HH-HHHHHEHH----HHHHHHHHHHHHHHHHHHHH-----
Results – PCR Amplification
PCR amplification of the peptide-coding regions from T4
genomic DNA (P1=Peptide 1, P2=Peptide 2)
Results – DNA purification
Agarose gel purification of the DNA fragment coding for
Peptide 2 (P2)
Results – Cloning
Testing for the presence of the right insert in the correct
orientation by PCR
Results – Induction
SDS-PAGE analysis of IPTG-induced protein expression
Results – Peptide Purification
SDS-PAGE analysis of the purification process for Peptide 2
Results – ATPase assay
ATPase assay to evaluate the activity of the truncated gp16
protein (Peptide 2)
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.
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
On a final note…
This presentation and the comprehensive
paper are available on-line at
http://www.karoly.tk
Any questions?

Thank you for your time!