1F0B

CRYSTAL STRUCTURE OF THE GREEN FLUORESCENT PROTEIN (GFP) VARIANT YFP-H148Q

1F0B の概要

• エントリーDOI: 10.2210/pdb1f0b/pdb
• 関連するPDBエントリー: 1F09 1YFP 2YFP
• 分子名称: GREEN FLUORESCENT PROTEIN (2 entities in total)
• 機能のキーワード: beta barrel, luminescence, bioluminescence, photoactive protein, green fluorescent protein, 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: 26951.41

構造登録者

Wachter, R.M.,Yarbrough, D.,Kallio, K.,Remington, S.J. (登録日: 2000-05-15, 公開日: 2000-11-17, 最終更新日: 2024-10-30)

主引用文献

Wachter, R.M.,Yarbrough, D.,Kallio, K.,Remington, S.J.
Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein.
J.Mol.Biol., 301:157-171, 2000

PubMed Abstract: The fluorescence emission of yellow fluorescent proteins (YFPs) has been shown to respond rapidly and reversibly to changes in the concentration of some small anions such as halides; this allows for the use of YFPs as genetically encodable Cl(-) sensors that may be targeted to specific organelles in living cells. Fluorescence is suppressed due to protonation of the chromophore upon anion binding, with a stronger level of interaction at low pH values. At pH 6.0, the apparent dissociation constant (K(app)) for Cl(-) is 32 mM for YFP and 22 mM for YFP-H148Q, whereas at pH 7.5, K(app) is 777 mM and 154 mM, respectively. In the cytosol, YFP-H148Q appears most promising as a halide sensor due to its high degree of sensitivity towards I(-) (K(app)=23 mM at pH 7.5). To aid in the design of variants with improved levels of specificity and affinity for Cl(-), we solved apo and I(-)-bound crystal structures of YFP-H148Q to 2.1 A resolution. The halide-binding site is found near van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stabilization. The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispensible for tight binding. A series of conformational changes occurs in the amphiphilic site upon anion binding, which appear to be propagated to the beta-bulge region around residue 148 on the protein surface. Anion binding raises the chromophore pK(a) values, since delocalization of the phenolate negative charge over the chromophore skeleton is suppressed. Extraction of microscopic binding constants for the linked equilibrium between anion and proton binding indicates that anion selectivity by YFP is related to hydration forces. Specific suggestions to improve Cl(-) binding to YFP-H148Q based on size and hydration energy are proposed.

PubMed: 10926499
DOI: 10.1006/jmbi.2000.3905

実験手法

X-RAY DIFFRACTION (2.1 Å)