3 research outputs found
Analytical gel filtration experiments to determine the MvaI oligomeric state and the stoichiometry of DNA binding for the oligoduplex 1 shown in ()
<p><b>Copyright information:</b></p><p>Taken from "Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically"</p><p></p><p>Nucleic Acids Research 2007;35(6):2035-2046.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874612.</p><p>© 2007 The Author(s)</p> Elution profiles were recorded simultaneously at 260 and 280 nm and deconvoluted to obtain separate curves for the MvaI (blue) and DNA (red) concentration. () MvaI alone, () DNA alone, () mixture with a 2:1 molar excess of MvaI over DNA, () stoichiometric mixture, () mixture with a 2:1 molar excess of DNA over MvaI, () calibration curve for Superose ™ 12 HR 10/30 column (Amersham Biosciences) with standards from Biorad (vitamin B-12, 1.35 kDa; myoglobin, 17 kDa; ovalbumin, 44kDa; IgG, 150 kDa and thyroglobin, 670 kDa)
Structural Basis for Efficient Chromophore Communication and Energy Transfer in a Constructed Didomain Protein Scaffold
The construction of useful functional biomolecular components
not
currently part of the natural repertoire is central to synthetic biology.
A new light-capturing ultra-high-efficiency energy transfer protein
scaffold has been constructed by coupling the chromophore centers
of two normally unrelated proteins: the autofluorescent protein enhanced
green fluorescent protein (EGFP) and the heme-binding electron transfer
protein cytochrome <i>b</i><sub>562</sub> (cyt <i>b</i><sub>562</sub>). Using a combinatorial domain insertion strategy,
a variant was isolated in which resonance energy transfer from the
donor EGFP to the acceptor cyt <i>b</i><sub>562</sub> was
close to 100% as evident by virtually full fluorescence quenching
on heme binding. The fluorescence signal of the variant was also sensitive
to the reactive oxygen species H<sub>2</sub>O<sub>2</sub>, with high
signal gain observed due to the release of heme. The structure of
oxidized holoprotein, determined to 2.75 Ã… resolution, revealed
that the two domains were arranged side-by-side in a V-shape conformation,
generating an interchromophore distance of ∼17 Å (14 Å
edge-to-edge). Critical to domain arrangement is the formation of
a molecular pivot point between the two domains as a result of different
linker sequence lengths at each domain junction and formation of a
predominantly polar interdomain interaction surface. The retrospective
structural analysis has provided an explanation for the basis of the
observed highly efficient energy transfer through chromophore arrangement
in the directly evolved protein scaffold and provides an insight into
the molecular principles by which to design new proteins with coupled
functions
Structural Basis for Efficient Chromophore Communication and Energy Transfer in a Constructed Didomain Protein Scaffold
The construction of useful functional biomolecular components
not
currently part of the natural repertoire is central to synthetic biology.
A new light-capturing ultra-high-efficiency energy transfer protein
scaffold has been constructed by coupling the chromophore centers
of two normally unrelated proteins: the autofluorescent protein enhanced
green fluorescent protein (EGFP) and the heme-binding electron transfer
protein cytochrome <i>b</i><sub>562</sub> (cyt <i>b</i><sub>562</sub>). Using a combinatorial domain insertion strategy,
a variant was isolated in which resonance energy transfer from the
donor EGFP to the acceptor cyt <i>b</i><sub>562</sub> was
close to 100% as evident by virtually full fluorescence quenching
on heme binding. The fluorescence signal of the variant was also sensitive
to the reactive oxygen species H<sub>2</sub>O<sub>2</sub>, with high
signal gain observed due to the release of heme. The structure of
oxidized holoprotein, determined to 2.75 Ã… resolution, revealed
that the two domains were arranged side-by-side in a V-shape conformation,
generating an interchromophore distance of ∼17 Å (14 Å
edge-to-edge). Critical to domain arrangement is the formation of
a molecular pivot point between the two domains as a result of different
linker sequence lengths at each domain junction and formation of a
predominantly polar interdomain interaction surface. The retrospective
structural analysis has provided an explanation for the basis of the
observed highly efficient energy transfer through chromophore arrangement
in the directly evolved protein scaffold and provides an insight into
the molecular principles by which to design new proteins with coupled
functions