Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), anAequorea victoriagreen fluorescent protein-like green-emitting fluorescent protein from the stony coralGalaxea fascicularis

Abstract

Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 A resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 A, alpha = 90.96, beta = 103.41, gamma = 101.79 degrees. The Matthews coefficient (V(M) = 2.10 A(3) Da(-1)) indicated that the crystal contained two mAG molecules per asymmetric unit.

Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties

“…The structure of eCGP123 will be solved by molecular replacement using the coordinates of a structure of mAG (89% identity excluding the 6ÂHis tag; Fig. 1; PDB entry 3adf; Ebisawa et al, 2009). In other experiments designed to characterize its optical properties, we have established that unlike the protein used to guide its design (mAG), eCGP123 possesses photoswitching properties similar to those of Dronpa (data not shown), a well characterized green photoswitching protein for which the crystal structure is known (Ando et al, 2004;Wilmann et al, 2006;Andresen et al, 2007).…”

In-situ and real-time growth observation of high-quality protein crystals under quasi-microgravity on earth

“…mKO protein was expressed in Escherichia coli and purified using a protocol similar to that used for the homologous fluorescent protein, monomeric Azami-Green 17 . Each subunit of the ZPαβ protein from Thermus thermophilus HB8 was co-expressed in E. coli Rosetta (DE3) (Novagen).…”

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

“…Four additional amino-acid substitutions (R149E, A160R, D182E and V191I) were found in mAG (Ebisawa et al, 2009) and the locations of these residues are distant from the QYG chromophore, suggesting that they do not affect the luminescent properties of mAG. Among these four additional amino-acid substitutions, the side chains of Glu182 and Ile191 do not face the probable tetrameric interfaces suggested from the locations of the three point mutations introduced to produce mAG.…”

“…Expression, purification, crystallization and X-ray diffraction data collection were performed as reported previously (Ebisawa et al, 2009). All of the programs used for structure determination and refinement are included in the CCP4 suite (Collaborative Computational Project, Number 4, 1994).…”

“…In addition, the side chain of Cys62 in the CYG 0 † R factor = P hkl jF obs j À jF calc j = P hkl jF obs j. R free was calculated using 5% of data that were excluded from refinement. ‡ This number corresponds to 95% of the number of unique reflections (20 674; Ebisawa et al, 2009). Table 2 The properties of fluorescent proteins structurally similar to mAG.…”