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PDBsum entry 2v6i
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References listed in PDB file
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Key reference
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Title
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Structure and biochemical analysis of kokobera virus helicase.
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Authors
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S.Speroni,
L.De colibus,
E.Mastrangelo,
E.Gould,
B.Coutard,
N.L.Forrester,
S.Blanc,
B.Canard,
A.Mattevi.
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Ref.
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Proteins, 2008,
70,
1120-1123.
[DOI no: ]
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PubMed id
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Abstract
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No abstract given.
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Figure 1.
Figure 1. (A) Helicase activity assay for wild-type (top) and
Met47Thr mutant (bottom). The substrate of the partially
double-stranded helicase assay[18] was obtained by annealing RNA
synthetic oligonucleotide whose sequence was designed to produce
a 16-base-pair duplex with a 14-nucleotide 3  overhand
in the longer strand (Primm, Milan-Italy). To form a
double-stranded substrate, the 5 -CACCUCUCUAGAGUCGACCUGCAGGCAUCG-3
strand
was labelled with [  -^32P]ATP
at its 5 end
by using T4 polynucleotide kinase, and annealed with the
complementary primer 5 -CGACUCUAGAGAGGUG-3
.
The annealed duplex was purified with Sephadex G25 columns
(GE-Healthcare). The assay was performed with 20  L
of the reaction buffer containing 25 mM Hepes pH 7.5, 1 mM
MgCl[2], 2 mM DTT, 2 mM ATP, 5% glycerol, 5U RNAsin for the RNA
substrate and 10 fmol of RNA substrate. The reaction was started
by adding the recombinant proteins at various concentrations
(50-1000 nM), or an equivalent volume of the buffer, and stopped
after 30 min at 37°C by adding 6 L
of loading dye (50% EDTA, 0.5% SDS, 50% glycerol, 0.01%
bromophenol blue). The helicase assay mixtures were resolved by
electrophoresis through nondenaturing 17% polyacrylamide gels
that were dried and analyzed by phosphoimage (Typhoon,
GE-Healthcare). The percentage of duplex unwinding was
calculated using the ImageQuant software (Amersham Bioscience)
by comparing the intensities of the two bands. (B) Ribbon
representation of Kokobera virus helicase. Domain 1 (residues
2-138), domain 2 (139-297), and domain 3 (297-431) are colored
orange, blue, and green, respectively. The bound pyrophosphate
ion is shown in ball-and-stick representation (oxygen in red and
phosphorous in green). Residues 60-68 are disordered and not
present in the refined model. (C) Close-up view of the ATPase
site and of Met47. The orientation and domain colors are as in
(B). Carbons are in yellow, nitrogen in blue, oxygen in red, and
phosphorous in green. Figures for (B) and (C) were generated
with Pymol (www.pymol.org).
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The above figure is
reprinted
by permission from John Wiley & Sons, Inc.:
Proteins
(2008,
70,
1120-1123)
copyright 2008.
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