6GO9

Structure of GFPmut2 crystallized at pH 6 and transferred to pH 7

6GO9 の概要

• エントリーDOI: 10.2210/pdb6go9/pdb
• 関連するPDBエントリー: 6GO8
• 分子名称: Green fluorescent protein, (4S)-2-METHYL-2,4-PENTANEDIOL, (4R)-2-METHYLPENTANE-2,4-DIOL, ... (4 entities in total)
• 機能のキーワード: beta barrel, bioluminescence, fluorescent 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: 28024.79

構造登録者

Lolli, G.,Raboni, S.,Pasqualetto, E.,Campanini, B.,Mozzarelli, A.,Bettati, S.,Battistutta, R. (登録日: 2018-06-01, 公開日: 2018-12-19, 最終更新日: 2024-01-17)

主引用文献

Lolli, G.,Raboni, S.,Pasqualetto, E.,Benoni, R.,Campanini, B.,Ronda, L.,Mozzarelli, A.,Bettati, S.,Battistutta, R.
Insight into GFPmut2 pH Dependence by Single Crystal Microspectrophotometry and X-ray Crystallography.
J.Phys.Chem.B, 122:11326-11337, 2018

PubMed Abstract: The fluorescence of Green Fluorescent Protein (wtGFP) and variants has been exploited in distinct applications in cellular and analytical biology. GFPs emission depends on the population of the protonated (A-state) and deprotonated (B-state) forms of the chromophore. Whereas wtGFP is pH-independent, mutants in which Ser65 is replaced by either threonine or alanine (as in GFPmut2) are pH-dependent, with a p K around 6. Given the wtGFP pH-independence, only the structure of the protonated form was determined. The deprotonated form was deduced on the basis of the crystal structure of the Ser65Thr mutant at basic pH, assuming that it corresponds to the conformation populated in solution. Here, we present an investigation where structures of the protonated and deprotonated forms of GFPmut2 were determined from crystals grown in either MPD at pH 6 or PEG at pH 8.5, and moved to either higher or lower pH. Both crystal forms of GFPmut2 were titrated monitoring the process via polarized absorption microspectrophotometry in order to precisely correlate the protonation process with the structures. We found that (i) in solution, chromophore titration is not thermodynamically coupled with any residue and Glu222 is always protonated independent of the protonation state of the chromophore; (ii) the lack of coupling is reflected in the structural behavior of the chromophore and Glu222 environments, with only the former showing variations with pH; (iii) titrations of low-pH and high-pH grown crystals exhibit a Hill coefficient of about 0.75, indicating an anticooperative behavior not observed in solution; (iv) structures where pH was changed in the crystal point to Glu222 as the ionizable group responsible for the outset of the anticooperative behavior; and (v) in GFPmut2 the canonical GFP proton wire involving the chromophore is not interrupted at the level of Ser205 and Glu222 at basic pH as in the Ser65Thr mutant. This allows proposing the structure of the deprotonated state of GFPmut2 as an alternative model for the analogous state of wtGFP.

PubMed: 30179482
DOI: 10.1021/acs.jpcb.8b07260

実験手法

X-RAY DIFFRACTION (1.672 Å)