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PDBsum entry 2awk
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Luminescent protein
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PDB id
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2awk
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References listed in PDB file
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Key reference
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Title
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Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis.
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Authors
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T.I.Wood,
D.P.Barondeau,
C.Hitomi,
C.J.Kassmann,
J.A.Tainer,
E.D.Getzoff.
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Ref.
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Biochemistry, 2005,
44,
16211-16220.
[DOI no: ]
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PubMed id
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Abstract
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Aequoria victoria green fluorescent protein (GFP) is a revolutionary molecular
biology tool because of its spontaneous peptide backbone cyclization and
chromophore formation from residues Ser65, Tyr66, and Gly67. Here we use
structure-based design, comprehensive targeted mutagenesis, and high-resolution
crystallography to probe the significant functional role of conserved Arg96
(R96) in chromophore maturation. The R96M GFP variant, in which the R96M side
chain is similar in volume but lacks the R96 positive charge, exhibits
dramatically slower chromophore maturation kinetics (from hours to months).
Comparison of the precyclized conformation of the chromophore-forming residues
with the mature R96M chromophore reveals a similar Y66 conformer, contrary to
the large Y66 conformational change previously defined in the slowly maturing
R96A variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A.,
and Getzoff, E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116].
Comprehensive R96 mutagenesis and fluorescent colony screening indicate that
only the R96K substitution restores wild-type maturation kinetics. Further, we
show that the slowly maturing R96A variant can be complemented with a Q183R
second-site mutation designed to restore the missing R96 positive charge and
rapid fluorophore biosynthesis. Moreover, comparative structural analysis of
R96M, R96K, R96A/Q183R, and wild-type GFP reveals the importance of the presence
of positive charge, rather than its exact position. Together, these structural,
mutational, and biochemical results establish a pivotal role for the R96
positive charge in accelerating the GFP post-translational modification, with
implications for peptide backbone cyclization in GFP, its homologues, and
related biological systems.
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