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PDBsum entry 1bfp
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References listed in PDB file
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Key reference
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Title
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Crystal structure and photodynamic behavior of the blue emission variant y66h/y145f of green fluorescent protein.
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Authors
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R.M.Wachter,
B.A.King,
R.Heim,
K.Kallio,
R.Y.Tsien,
S.G.Boxer,
S.J.Remington.
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Ref.
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Biochemistry, 1997,
36,
9759-9765.
[DOI no: ]
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PubMed id
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Abstract
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The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea
victoria green fluorescent protein has been determined by molecular replacement
and the model refined. The crystallographic R-factor is 18.1% for all data from
20 to 2.1 A, and the model geometry is excellent. The chromophore is non-native
and is autocatalytically generated from the internal tripeptide
Ser65-His66-Gly67. The final electron density maps indicate that the formation
of the chromophore is complete, including 1,2 dehydration of His66 as indicated
by the planarity of the chromophore. The chromophore is in the cis conformation,
with no evidence for any substantial fraction of the trans configuration or
uncyclized apoprotein, and is well-shielded from bulk solvent by the folded
protein. These characteristics indicate that the machinery for production of the
chromophore from a buried tripeptide unit is not only intact but also highly
efficient in spite of a major change in chromophore chemical structure.
Nevertheless, there are significant rearrangements in the hydrogen bond
configuration around the chromophore as compared to wild-type, indicating
flexibility of the active site. pH titration of the intact protein and the
chromopeptide (pKa1 = 4.9 +/- 0.1, pKa2 = 12.0 +/- 0.1) suggests that the
predominant form of the chromophore in the intact protein is electrically
neutral. In contrast to the wild-type protein [Chattoraj, M., King, B. A.,
Bublitz, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A.,
8362-8367], femtosecond fluorescence up-conversion spectroscopy of the intact
protein and a partially deuterated form strongly suggests that excited-state
proton transfer is not coupled to fluorescence emission.
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Secondary reference #1
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Title
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Engineering green fluorescent protein for improved brightness, Longer wavelengths and fluorescence resonance energy transfer.
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Authors
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R.Heim,
R.Y.Tsien.
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Ref.
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Curr Biol, 1996,
6,
178-182.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Fluorescence excitation and emission spectra of
different GFP mutants. All spectra were normalized to a maximal
value of 1. Each pair of excitation and emission spectra is
depicted by a distinct line colour.
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Figure 3.
Figure 3. Energy transfer between covalently linked BFP and
GFP is abolished after cleavage with trypsin. The green GFP
mutant S65C [5] was linked to the blue mutant Y66H/Y145F by
expressing their fused cDNAs with a linker sequence that can be
cleaved by trypsin or enterokinase. Excited at 368 nm, the
uncleaved dimer emitted bright green light that gradually dimmed
upon cleavage of the linker to separate the protein domains. As
the cleavage by trypsin progressed (0, 2, 5, 10 and 47 min),
more blue light was emitted. There was no further change after
47 min. Similar results were obtained with enterokinase cleavage
(data not shown).
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The above figures are
reproduced from the cited reference
with permission from Cell Press
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