1KYP

Crystal Structure of an Apo Green Fluorescent Protein Zn Biosensor

1KYP の概要

• エントリーDOI: 10.2210/pdb1kyp/pdb
• 関連するPDBエントリー: 1kyr 1kys
• 分子名称: Green Fluorescent Protein, MAGNESIUM ION (3 entities in total)
• 機能のキーワード: beta barrel, chromophore, apo structure, luminescent protein
• 由来する生物種: Aequorea victoria
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 26736.31

構造登録者

Barondeau, D.P.,Kassmann, C.J.,Tainer, J.A.,Getzoff, E.D. (登録日: 2002-02-05, 公開日: 2002-04-10, 最終更新日: 2024-11-13)

主引用文献

Barondeau, D.P.,Kassmann, C.J.,Tainer, J.A.,Getzoff, E.D.
Structural chemistry of a green fluorescent protein Zn biosensor.
J.Am.Chem.Soc., 124:3522-3524, 2002

PubMed Abstract: 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.

PubMed: 11929238
DOI: 10.1021/ja0176954

実験手法

X-RAY DIFFRACTION (1.35 Å)