The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission

Abstract

PDB Reference: monomeric mutant of AzamiGreen, 3adf.Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants.

CYK4 Promotes Antiparallel Microtubule Bundling by Optimizing MKLP1 Neck Conformation

“…We started by observing full-length C . elegans ZEN-4 kinesin tagged with a green fluorescent protein, monomeric Azami Green (Z775mAG, Fig 1A and 1B ) [ 31 , 32 ], adsorbed onto a mica surface. Under a salt free condition ( Fig 2A , S1 Movie ), which would promote adsorption of the protein to the mica surface and thus allow a better resolution, we observed that the tagged ZEN-4 molecule consists of a pair of globular domains (pseudocolored in yellow in the bottom rows), a nearby smaller globular mass (cyan), a short rod-like structure (magenta), flexible linkers (magenta), and another pair of fluctuating globular domains (green).…”

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure‐guided surface engineering

“…(C)]. Critical residues for proper chromophore coordination and fluorescence include Arg66, Ser142, and His193, and are invariant with respect to similar proteins such as monomeric Azami Green (mAG) and Dronpa . Maintaining planarity and rigidity of the 4‐hydroxylbenzyl ring (Tyr63) relative to the imidazoline ring (Gln62, Gly64) of the chromophore is important for fluorescence, and its position is coordinated by a network of residues that line the chromophore cavity (Supporting Information Table S1).…”

“…Indexing and scaling using Mosflm of the data in a monoclinic (or higher) Laue group indicated the data was consistent with space group P1 and POINTLESS was used to confirm the correct Laue and space group. The eCGP123 structure was solved using molecular replacement and mAG (PDB entry: 3adf) as a search ensemble. Crystals of TGP were grown using the hanging drop vapor diffusion method where 3 µL of purified protein (27 mg/mL in 20 m M Hepes pH 7.4 100 m M NaCl) was mixed with 3 µL of well solution (0.1 M Tris‐HCl pH 7.0 and 20% w/v Polyethylene glycol monomethyl ether 2000).…”

Structural consequences of chromophore formation and exploration of conserved lid residues amongst naturally occurring fluorescent proteins

“…The coordinates of the crystal structures of all the wild-type GFP-like proteins were obtained from the Protein Data Bank (PDB)(31) - (1GFL(32), 1MOU(33), 1UIS(34), 1XSS(35), 1YZW(36), 1ZGO(37), 1ZUX(38), 2A46(39), 2C9I(40), 2C9J(41), 2DD7(42), 2G3O(36), 2GW3(43), 2IB5(44), 2ICR(45), 2IE2(46), 2OGR(47), 2OJK(45), 2RH7(48), 2WHT(49), 2Z6X(50), 2ZMU(51), 3CGL(52), 3GB3(53), 3H1O(54), 3MGF(55), 3PIB(53), and 3PJ5(53)). The protein preparation workflow(56) and Epik v2.0109(57) were used with hydrogen bond optimization to add hydrogen atoms to protein and solvent atoms as required.…”