2hqk
From Proteopedia
(New page: 200px<br /><applet load="2hqk" size="350" color="white" frame="true" align="right" spinBox="true" caption="2hqk, resolution 1.19Å" /> '''Structure of a monom...) |
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==Overview== | ==Overview== | ||
| - | The arsenal of engineered variants of the GFP [green FP (fluorescent | + | The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor. |
==About this Structure== | ==About this Structure== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Ai, H.]] | [[Category: Ai, H.]] | ||
| - | [[Category: Campbell, R | + | [[Category: Campbell, R E.]] |
| - | [[Category: Henderson, J | + | [[Category: Henderson, J N.]] |
| - | [[Category: Remington, S | + | [[Category: Remington, S J.]] |
[[Category: ACT]] | [[Category: ACT]] | ||
[[Category: CL]] | [[Category: CL]] | ||
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[[Category: 11-stranded beta barrel]] | [[Category: 11-stranded beta barrel]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:44:37 2008'' |
Revision as of 15:44, 21 February 2008
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Structure of a monomeric cyan fluorescent protein derived from Clavularia
Overview
The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.
About this Structure
2HQK is a Single protein structure of sequence from Clavularia sp. with , and as ligands. Full crystallographic information is available from OCA.
Reference
Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging., Ai HW, Henderson JN, Remington SJ, Campbell RE, Biochem J. 2006 Dec 15;400(3):531-40. PMID:16859491
Page seeded by OCA on Thu Feb 21 17:44:37 2008
