1kys
From Proteopedia
(New page: 200px<br /> <applet load="1kys" size="450" color="white" frame="true" align="right" spinBox="true" caption="1kys, resolution 1.44Å" /> '''Crystal Structure o...) |
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caption="1kys, resolution 1.44Å" /> | caption="1kys, resolution 1.44Å" /> | ||
'''Crystal Structure of a Zn-bound Green Fluorescent Protein Biosensor'''<br /> | '''Crystal Structure of a Zn-bound Green Fluorescent Protein Biosensor'''<br /> | ||
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| + | ==Overview== | ||
| + | We designed a green fluorescent protein mutant (BFPms1) that, preferentially binds Zn(II) (enhancing fluorescence intensity) and Cu(II), (quenching fluorescence) directly to a chromophore ligand that resembles a, dipyrrole unit of a porphyrin. Crystallographic structure determination of, apo, Zn(II)-bound, and Cu(II)-bound BFPms1 to better than 1.5 A resolution, allowed us to refine metal centers without geometric restraints, to, calculate experimental standard uncertainty errors for bond lengths and, angles, and to model thermal displacement parameters anisotropically. The, BFPms1 Zn(II) site (KD = 50 muM) displays distorted trigonal bipyrimidal, geometry, with Zn(II) binding to Glu222, to a water molecule, and, tridentate to the chromophore ligand. In contrast, the BFPms1 Cu(II) site, (KD = 24 muM) exhibits square planar geometry similar to metalated, porphyrins, with Cu(II) binding to the chromophore chelate and Glu222. The, apo structure reveals a large electropositive region near the designed, metal insertion channel, suggesting a basis for the measured metal cation, binding kinetics. The preorganized tridentate ligand is accommodated in, both coordination geometries by a 0.4 A difference between the Zn and Cu, positions and by distinct rearrangements of Glu222. The highly accurate, metal ligand bond lengths reveal different protonation states for the same, oxygen bound to Zn vs Cu, with implications for the observed metal ion, specificity. Crystallographic anisotropic thermal factor analysis, validates metal ion rigidification of the chromophore in enhancement of, fluorescence intensity upon Zn(II) binding. Thus, our high-resolution, structures reveal how structure-based design has effectively linked, selective metal binding to changes in fluorescent properties. Furthermore, this protein Zn(II) biosensor provides a prototype suitable for further, optimization by directed evolution to generate metalloprotein variants, with desirable physical or biochemical properties. | ||
==About this Structure== | ==About this Structure== | ||
| - | 1KYS 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 ZN as [http://en.wikipedia.org/wiki/ligand ligand]. The following page contains interesting information on the relation of 1KYS with [[http://pdb.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb42_1.html Green Fluorescent Protein (GFP)]]. Full crystallographic information is available from [http:// | + | 1KYS 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=ZN:'>ZN</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. The following page contains interesting information on the relation of 1KYS with [[http://pdb.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb42_1.html Green Fluorescent Protein (GFP)]]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KYS OCA]. |
==Reference== | ==Reference== | ||
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[[Category: zn binding design]] | [[Category: zn binding design]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri Feb 15 16:15:44 2008'' |
Revision as of 14:15, 15 February 2008
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Crystal Structure of a Zn-bound Green Fluorescent Protein Biosensor
Overview
We designed a green fluorescent protein mutant (BFPms1) that, preferentially binds Zn(II) (enhancing fluorescence intensity) and Cu(II), (quenching fluorescence) directly to a chromophore ligand that resembles a, dipyrrole unit of a porphyrin. Crystallographic structure determination of, apo, Zn(II)-bound, and Cu(II)-bound BFPms1 to better than 1.5 A resolution, allowed us to refine metal centers without geometric restraints, to, calculate experimental standard uncertainty errors for bond lengths and, angles, and to model thermal displacement parameters anisotropically. The, BFPms1 Zn(II) site (KD = 50 muM) displays distorted trigonal bipyrimidal, geometry, with Zn(II) binding to Glu222, to a water molecule, and, tridentate to the chromophore ligand. In contrast, the BFPms1 Cu(II) site, (KD = 24 muM) exhibits square planar geometry similar to metalated, porphyrins, with Cu(II) binding to the chromophore chelate and Glu222. The, apo structure reveals a large electropositive region near the designed, metal insertion channel, suggesting a basis for the measured metal cation, binding kinetics. The preorganized tridentate ligand is accommodated in, both coordination geometries by a 0.4 A difference between the Zn and Cu, positions and by distinct rearrangements of Glu222. The highly accurate, metal ligand bond lengths reveal different protonation states for the same, oxygen bound to Zn vs Cu, with implications for the observed metal ion, specificity. Crystallographic anisotropic thermal factor analysis, validates metal ion rigidification of the chromophore in enhancement of, fluorescence intensity upon Zn(II) binding. Thus, our high-resolution, structures reveal how structure-based design has effectively linked, selective metal binding to changes in fluorescent properties. Furthermore, this protein Zn(II) biosensor provides a prototype suitable for further, optimization by directed evolution to generate metalloprotein variants, with desirable physical or biochemical properties.
About this Structure
1KYS is a Single protein structure of sequence from Aequorea victoria with as ligand. The following page contains interesting information on the relation of 1KYS with [Green Fluorescent Protein (GFP)]. Full crystallographic information is available from OCA.
Reference
Structural chemistry of a green fluorescent protein Zn biosensor., Barondeau DP, Kassmann CJ, Tainer JA, Getzoff ED, J Am Chem Soc. 2002 Apr 10;124(14):3522-4. PMID:11929238
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