2yfp: Difference between revisions

Revision as of 19:57, 21 February 2008

STRUCTURE OF YELLOW-EMISSION VARIANT OF GFP

OverviewOverview

BACKGROUND: Because of its ability to spontaneously generate its own fluorophore, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria is used extensively as a fluorescent marker in molecular and cell biology. The yellow fluorescent proteins (YFPs) have the longest wavelength emissions of all GFP variants examined to date. This shift in the spectrum is the result of a T203Y substitution (single-letter amino acid code), a mutation rationally designed on the basis of the X-ray structure of GFP S65T. RESULTS: We have determined the crystal structures of YFP T203Y/S65G/V68L/S72A and YFP H148G to 2.5 and 2.6 A resolution, respectively. Both structures show clear electron density for nearly coplanar pi-pi stacking between Tyr203 and the chromophore. The chromophore has been displaced by nearly 1 A in comparison to other available structures. Although the H148G mutation results in the generation of a solvent channel to the chromophore cavity, intense fluorescence is maintained. The chromophore in the intact protein can be titrated, and the two variants have pKa values of 7.0 (YFP) and 8.0 (YFP H148G). CONCLUSIONS: The observed red shift of the T203Y YFP variant is proposed to be mainly due to the additional polarizability of the pi-stacked Tyr203. The altered location of the chromophore suggests that the exact positions of nearby residues are not crucial for the chemistry of chromophore formation. The YFPs significantly extend the pH range over which GFPs may be employed as pH indicators in live cells.

About this StructureAbout this Structure

2YFP is a Single protein structure of sequence from Aequorea victoria. Full crystallographic information is available from OCA.

ReferenceReference

Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein., Wachter RM, Elsliger MA, Kallio K, Hanson GT, Remington SJ, Structure. 1998 Oct 15;6(10):1267-77. PMID:9782051

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 ==Overview==
-BACKGROUND: Because of its ability to spontaneously generate its own, fluorophore, the green fluorescent protein (GFP) from the jellyfish, Aequorea victoria is used extensively as a fluorescent marker in molecular, and cell biology. The yellow fluorescent proteins (YFPs) have the longest, wavelength emissions of all GFP variants examined to date. This shift in, the spectrum is the result of a T203Y substitution (single-letter amino, acid code), a mutation rationally designed on the basis of the X-ray, structure of GFP S65T. RESULTS: We have determined the crystal structures, of YFP T203Y/S65G/V68L/S72A and YFP H148G to 2.5 and 2.6 A resolution, respectively. Both structures show clear electron density for nearly, coplanar pi-pi stacking between Tyr203 and the chromophore. The, chromophore has been displaced by nearly 1 A in comparison to other, available structures. Although the H148G mutation results in the, generation of a solvent channel to the chromophore cavity, intense, fluorescence is maintained. The chromophore in the intact protein can be, titrated, and the two variants have pKa values of 7.0 (YFP) and 8.0 (YFP, H148G). CONCLUSIONS: The observed red shift of the T203Y YFP variant is, proposed to be mainly due to the additional polarizability of the, pi-stacked Tyr203. The altered location of the chromophore suggests that, the exact positions of nearby residues are not crucial for the chemistry, of chromophore formation. The YFPs significantly extend the pH range over, which GFPs may be employed as pH indicators in live cells.
+BACKGROUND: Because of its ability to spontaneously generate its own fluorophore, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria is used extensively as a fluorescent marker in molecular and cell biology. The yellow fluorescent proteins (YFPs) have the longest wavelength emissions of all GFP variants examined to date. This shift in the spectrum is the result of a T203Y substitution (single-letter amino acid code), a mutation rationally designed on the basis of the X-ray structure of GFP S65T. RESULTS: We have determined the crystal structures of YFP T203Y/S65G/V68L/S72A and YFP H148G to 2.5 and 2.6 A resolution, respectively. Both structures show clear electron density for nearly coplanar pi-pi stacking between Tyr203 and the chromophore. The chromophore has been displaced by nearly 1 A in comparison to other available structures. Although the H148G mutation results in the generation of a solvent channel to the chromophore cavity, intense fluorescence is maintained. The chromophore in the intact protein can be titrated, and the two variants have pKa values of 7.0 (YFP) and 8.0 (YFP H148G). CONCLUSIONS: The observed red shift of the T203Y YFP variant is proposed to be mainly due to the additional polarizability of the pi-stacked Tyr203. The altered location of the chromophore suggests that the exact positions of nearby residues are not crucial for the chemistry of chromophore formation. The YFPs significantly extend the pH range over which GFPs may be employed as pH indicators in live cells.
 ==About this Structure==
@@ Line 13: / Line 13: @@
 [[Category: Aequorea victoria]]
 [[Category: Single protein]]
-[[Category: Elsliger, M.A.]]
+[[Category: Elsliger, M A.]]
-[[Category: Hanson, G.T.]]
+[[Category: Hanson, G T.]]
 [[Category: Kallio, K.]]
-[[Category: Remington, S.J.]]
+[[Category: Remington, S J.]]
-[[Category: Wachter, R.M.]]
+[[Category: Wachter, R M.]]
 [[Category: bioluminescence]]
 [[Category: fluorescent tag]]
@@ Line 24: / Line 24: @@
 [[Category: yellow-emission variant]]
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jan 29 21:32:58 2008''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:57:01 2008''