2awm
From Proteopedia
(New page: 200px<br /><applet load="2awm" size="450" color="white" frame="true" align="right" spinBox="true" caption="2awm, resolution 1.70Å" /> '''GFP R96A chromophore...) |
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| - | [[Image:2awm.gif|left|200px]]<br /><applet load="2awm" size=" | + | [[Image:2awm.gif|left|200px]]<br /><applet load="2awm" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="2awm, resolution 1.70Å" /> | caption="2awm, resolution 1.70Å" /> | ||
'''GFP R96A chromophore maturation recovery mutant R96A Q183R'''<br /> | '''GFP R96A chromophore maturation recovery mutant R96A Q183R'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Aequoria victoria green fluorescent protein (GFP) is a revolutionary | + | 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. |
==About this Structure== | ==About this Structure== | ||
| - | 2AWM is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Aequorea_victoria Aequorea victoria] with MG as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http:// | + | 2AWM is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Aequorea_victoria Aequorea victoria] with <scene name='pdbligand=MG:'>MG</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2AWM OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Aequorea victoria]] | [[Category: Aequorea victoria]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Barondeau, D | + | [[Category: Barondeau, D P.]] |
| - | [[Category: Getzoff, E | + | [[Category: Getzoff, E D.]] |
[[Category: Hitomi, C.]] | [[Category: Hitomi, C.]] | ||
| - | [[Category: Kassmann, C | + | [[Category: Kassmann, C J.]] |
| - | [[Category: Tainer, J | + | [[Category: Tainer, J A.]] |
| - | [[Category: Wood, T | + | [[Category: Wood, T I.]] |
[[Category: MG]] | [[Category: MG]] | ||
[[Category: barrel]] | [[Category: barrel]] | ||
[[Category: gfp chromophore recovery mutant r96a q183r]] | [[Category: gfp chromophore recovery mutant r96a q183r]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:31:43 2008'' |
Revision as of 14:31, 21 February 2008
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GFP R96A chromophore maturation recovery mutant R96A Q183R
Overview
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.
About this Structure
2AWM is a Single protein structure of sequence from Aequorea victoria with as ligand. Full crystallographic information is available from OCA.
Reference
Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis., Wood TI, Barondeau DP, Hitomi C, Kassmann CJ, Tainer JA, Getzoff ED, Biochemistry. 2005 Dec 13;44(49):16211-20. PMID:16331981
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