Abstract
Green fluorescent protein (GFP) is a light-emitting protein that does not require a prosthetic group for its fluorescent activity. As such, GFP has become indispensable as a molecular tool in molecular biology. Nonetheless, there has been no subatomic elucidation of the GFP structure owing to the structural polymorphism around the chromophore. Here, subatomic resolution X-ray structures of GFP without the structural polymorphism are reported. The positions of H atoms, hydrogen-bonding network patterns and accurate geometric parameters were determined for the two protonated forms. Compared with previously determined crystal structures and theoretically optimized structures, the anionic chromophores of the structures represent the authentic resonance state of GFP. In addition, charge-density analysis based on atoms-in-molecules theory and noncovalent interaction analysis highlight weak but substantial interactions between the chromophore and the protein environment. Considered with the derived chemical indicators, the lone pair-π interactions between the chromophore and Thr62 should play a sufficient role in maintaining the electronic state of the chromophore. These results not only reveal the fine structural features that are critical to understanding the properties of GFP, but also highlight the limitations of current quantum-chemical calculations.
Highlights
Green fluorescent protein (GFP), which was discovered in the jellyfish Aequorea victoria, is a light-emitting protein consisting of 238 amino acids
The quantum-chemical calculations are strongly biased by the initial geometry, while the crystal structures are used after energy optimization
PDB entry 1ema, which is one of the first crystal structures of GFP (Ormoet al., 1996), has a C—O bond length of 1.48 A. This value is much longer than the lengths in the other structures shown in Fig. 4, while the crystal structure has been used as an initial model in some theoretical calculations of the chromophore
Summary
Green fluorescent protein (GFP), which was discovered in the jellyfish Aequorea victoria, is a light-emitting protein consisting of 238 amino acids. GFP absorbs UV–blue light and emits green light. The structure consists of an 11-stranded -barrel plugged by a chromophore [Fig. 1(a)]. There are dual protonation states of the hydroxybenzyl (tyrosyl) group of the chromophore in the protein. The protonated neutral ‘A’ and deprotonated anionic ‘B’ forms [Fig. 1(b)] are present at ratios of 6:1 to 4:1 in wildtype GFP (Brejc et al, 1997; Chattoraj et al, 1996). The two forms have distinct spectroscopic properties (Chattoraj et al, 1996). The absorption peak at 398 nm belongs to the A-form chromophore, while the peak at 475 nm belongs to the B form [Supplementary Fig. S1(a)]
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