1f98: Difference between revisions

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CRYSTAL STRUCTURE OF THE PHOTOACTIVE YELLOW PROTEIN MUTANT T50V

OverviewOverview

To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination. The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively. Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy. The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation. The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.

About this StructureAbout this Structure

1F98 is a Single protein structure of sequence from Halorhodospira halophila with as ligand. Full crystallographic information is available from OCA.

ReferenceReference

Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein., Brudler R, Meyer TE, Genick UK, Devanathan S, Woo TT, Millar DP, Gerwert K, Cusanovich MA, Tollin G, Getzoff ED, Biochemistry. 2000 Nov 7;39(44):13478-86. PMID:11063584

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-[[Image:1f98.jpg|left|200px]]<br /><applet load="1f98" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1f98.jpg|left|200px]]<br /><applet load="1f98" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1f98, resolution 1.15&Aring;" />
 '''CRYSTAL STRUCTURE OF THE PHOTOACTIVE YELLOW PROTEIN MUTANT T50V'''<br />
 ==Overview==
-To understand in atomic detail how a chromophore and a protein interact to, sense light and send a biological signal, we are characterizing, photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light, receptor which undergoes a photocycle upon illumination. The active site, residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form, a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69, chromophore in the PYP ground state, suggesting an essential role for, these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein, stability. Here, we describe the role of T50 and Y42 by use of, site-specific mutants. T50 and Y42 are involved in fine-tuning the, chromophore's absorption maximum. The high-resolution X-ray structures, show that the hydrogen-bonding interactions between the protein and the, chromophore are weakened in the mutants, leading to increased electron, density on the chromophore's aromatic ring and consequently to a red shift, of its absorption maximum from 446 nm to 457 and 458 nm in the mutants, T50V and Y42F, respectively. Both mutants have slightly perturbed, photocycle kinetics and, similar to the R52A mutant, are bleached more, rapidly and recover more slowly than the wild type. The effect of pH on, the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are, not directly involved in any protonation or deprotonation events that, control the speed of the light cycle. The unfolding energies, 26.8 and, 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared, to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced, protein stability gives rise to a second PYP population with an altered, chromophore conformation as shown by UV/visible and FT Raman spectroscopy., The second chromophore conformation gives rise to a shoulder at 391 nm in, the UV/visible absorption spectrum and indicates that the hydrogen bond, between Y42 and the chromophore is crucial for the stabilization of the, native chromophore and protein conformation. The two conformations in the, Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and, the acid titration curves suggest that the 391 nm form of the chromophore, is not fully protonated. The fluorescence quantum yield of the mutant Y42F, is 1.8% and is increased by an order of magnitude when compared to the, wild type.
+To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination. The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively. Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy. The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation. The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.
 ==About this Structure==
-F98 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Halorhodospira_halophila Halorhodospira halophila] with HC4 as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1F98 OCA].
+F98 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Halorhodospira_halophila Halorhodospira halophila] with <scene name='pdbligand=HC4:'>HC4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1F98 OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Single protein]]
 [[Category: Brudler, R.]]
-[[Category: Genick, U.K.]]
+[[Category: Genick, U K.]]
-[[Category: Getzoff, E.D.]]
+[[Category: Getzoff, E D.]]
-[[Category: Meyer, T.E.]]
+[[Category: Meyer, T E.]]
 [[Category: Tollin, G.]]
 [[Category: HC4]]
@@ Line 22: / Line 22: @@
 [[Category: photoreceptor]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 14:43:38 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:36:12 2008''