Sandbox 206: Difference between revisions
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== A new YFP called Venus (1MYW) == | == A new YFP called Venus (1MYW) == | ||
=== Introduction=== | === Introduction=== | ||
Yellow Fluorescent protein (YFP) is a variant of Green fluorescent protein (GFP). This protein is very useful like a biomarker in a lot of biological applications. First, the GFP was mutated In YFP. But the YFP had a low fluorescence because of a slow maturation and it was very sensitive to experimental condition (pH, Cl-). To increase the brightness of the protein, maturation speed and other properties, five mutations were done to obtain Venus, a Yellow Fluorescent Protein less sensitive to experimental condition. | Yellow Fluorescent protein (YFP) is a variant of Green fluorescent protein (GFP). A lot of search was made of this protein in order to improve his fluorescence, his viability in different middle and others conditions<ref> Improved green fluorescence; Heim R., Cubitt A.B. and Tsien R.Y; Nature 373, 663 - 664 (23 February 1995) Pubmed : [http://www.ncbi.nlm.nih.gov/pubmed/7854443 7854443] DOI : [http://www.nature.com/nature/journal/v373/n6516/abs/373663b0.html 10.1038/373663b0] Disponible on [http://tsienlab.ucsd.edu/Publications/Heim%201995%20Nature%20-%20Improved%20GFP.PDF http://tsienlab.ucsd.edu/Publications/Heim%201995%20Nature%20-%20Improved%20GFP.PDF]</ref>. This protein is very useful like a biomarker in a lot of biological applications. First, the GFP was mutated In YFP. But the YFP had a low fluorescence because of a slow maturation and it was very sensitive to experimental condition (pH, Cl-)<ref>1H, 15N and 13C assignments of yellow fluorescent protein (YFP) Venus; Hsu ST, Behrens C, Cabrita LD, Dobson CM.; Biomol NMR Assign. 2009 Jun;3(1):67-72. Epub 2009 Jan 13 Pubmed : [http://www.ncbi.nlm.nih.gov/pubmed/19636949 19636949] DOI : [http://www.citeulike.org/article/3897162 doi:10.1007/s12104-009-9143-y] Disponible on [https://springerlink3.metapress.com/content/k45571u5l6341v44/resource-secured/?target=fulltext.pdf&sid=dfpkgoxtq2cck1regroonku4&sh=www.springerlink.com https://springerlink3.metapress.com]</ref>. To increase the brightness of the protein, maturation speed and other properties, five mutations were done to obtain Venus, a Yellow Fluorescent Protein less sensitive to experimental condition. | ||
=== Presentation of the molecule === | === Presentation of the molecule === | ||
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==== Primary and Secondary structure ==== | ==== Primary and Secondary structure ==== | ||
Venus is a 27 kDa protein (26 875,50Da) consisting of 239 residues and form a secondary structure of 7 <scene name='Sandbox_206/Alpha_helix/1'>α-helix</scene> (2 in the center of the molecule) and 11 <scene name='Sandbox_206/Sheet/2'> β-sheet</scene>. This molecule is monomeric. Venus has many common point with the GFP and also with the EYFP. In all three structures compared, the cross-section taken perpendicularly to the long axis of the barrel is not perfectly circular but rather <scene name='Sandbox_206/Oval/1'>oval</scene> in shape. While the longer diameter of the oval cross-section of all three proteins is identical (23.8 Å measured from distances of four Cα pairs), the shorter diameter differs between EYFP (22.3 Å) and Venus (21.9 Å). The difference in the oval shape between these yellow variants is closely related to the packing of | Venus is a 27 kDa protein (26 875,50Da) consisting of 239 residues and form a secondary structure of 7 <scene name='Sandbox_206/Alpha_helix/1'>α-helix</scene> (2 in the center of the molecule) and 11 <scene name='Sandbox_206/Sheet/2'> β-sheet</scene>. This molecule is monomeric. Venus has many common point with the GFP and also with the EYFP. In all three structures compared, the cross-section taken perpendicularly to the long axis of the barrel is not perfectly circular but rather <scene name='Sandbox_206/Oval/1'>oval</scene> in shape. While the longer diameter of the oval cross-section of all three proteins is identical (23.8 Å measured from distances of four Cα pairs), the shorter diameter differs between EYFP (22.3 Å) and Venus (21.9 Å). The difference in the oval shape between these yellow variants is closely related to the packing of interior amino acid residues and influences the integrity of dimer interface<ref> Crystal Structure of Venus, a Yellow Fluorescent Protein with Improved Maturation and Reduced Environmental Sensitivity; Rekas, A., Alattia, J.R., Nagai, T., Miyawaki, A., and Ikura M., 2002 Pubmed : [http://www.ncbi.nlm.nih.gov/pubmed/12370172?dopt=Abstract 12370172] DOI : [http://www.jbc.org/content/277/52/50573 10.1074/jbc.M209524200] Disponible on [http://www.ebi.ac.uk/pdbe-srv/view/entry/1myw/citation.html]</ref>. | ||
==== The Chromophore ==== | ==== The Chromophore ==== | ||
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The residues surrounding the Venus <scene name='Sandbox_206/Chromophore/1'> chromophore </scene> are similar to those surrounding the chromophore of EYFP. Electron density studies of Venus show an 11° angle between the planes of the chromophore and Tyr203, while the same angle measured in EYFP is 11.5–12.3°, making the plane of the Venus chromophore slightly more parallel than that of EYFP. | The residues surrounding the Venus <scene name='Sandbox_206/Chromophore/1'> chromophore </scene> are similar to those surrounding the chromophore of EYFP. Electron density studies of Venus show an 11° angle between the planes of the chromophore and Tyr203, while the same angle measured in EYFP is 11.5–12.3°, making the plane of the Venus chromophore slightly more parallel than that of EYFP. | ||
[[Image:Chromophore1MYW.gif |350px|left|thumb|Chromophore of Venus]] | |||
[[Image:Chromophore1MYW.gif | | |||
This small difference may account for a minor change in the absorption spectrum of Venus rela- tive to EYFP, as seen at a pH range of 4.6–8.6 and a 50 mM NaCl concentration. Although the position of the main absorp- tion maximum remains unchanged (516 nm) in both Venus and EYFP, a smaller peak resulting from the neutral (protonated) chromophore is shifted in Venus by 20 nm producing a peak at 413 nm versus 393 nm in EYFP. The slightly more parallel orientation of the aromatic rings of Tyr66 and Tyr203 might also help improve the π-π interaction, thus reducing the excited state energy of the neutral chromophore. If this is the case, we can hypothesize that at lower pH, which is favorable to chro- mophore protonation, the angle between the rings would be smaller than 11°. | This small difference may account for a minor change in the absorption spectrum of Venus rela- tive to EYFP, as seen at a pH range of 4.6–8.6 and a 50 mM NaCl concentration. Although the position of the main absorp- tion maximum remains unchanged (516 nm) in both Venus and EYFP, a smaller peak resulting from the neutral (protonated) chromophore is shifted in Venus by 20 nm producing a peak at 413 nm versus 393 nm in EYFP. The slightly more parallel orientation of the aromatic rings of Tyr66 and Tyr203 might also help improve the π-π interaction, thus reducing the excited state energy of the neutral chromophore. If this is the case, we can hypothesize that at lower pH, which is favorable to chro- mophore protonation, the angle between the rings would be smaller than 11°. | ||
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Nikon : [http://www.microscopyu.com/print/articles/livecellimaging/fpintro-print.html http://www.microscopyu.com/print/articles/livecellimaging/fpintro-print.html] | Nikon : [http://www.microscopyu.com/print/articles/livecellimaging/fpintro-print.html http://www.microscopyu.com/print/articles/livecellimaging/fpintro-print.html] | ||
Introduction to Fluorescent Proteins : [http://www.microscopyu.com/articles/livecellimaging/fpintro.html http://www.microscopyu.com/articles/livecellimaging/fpintro.html] | |||
IGEM 2008 Uni Freiburg : [http://2008.igem.org/Team:Freiburg http://2008.igem.org/Team:Freiburg] | |||
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