Structural Analysis Of Yellow Fluorescent Protein MAmetrine With A Large Stokes Shift (LSS)

mAmetrine is a yellow fluorescent protein (YFP) with large Stokes shift (LSS). LSS is an accepted nomenclature in the field of fluorescent proteins to denote fluorescent proteins exhibiting excitation and emission maxima that differ greater than ¹00 nm. mAmetrine retains the property of violet excitation (excitation wavelength,λ_ex= 406 nm), and yellow emission (emission wavelength,λ_em= 526 nm) resulting from excited-state proton transfer (ESPT) andπ-πstacking interactions with the chromophore. However, the mechanism of ESPT for mAmetrine LSS fluorescence emission is unknown. To better understand mAmetrine function and study ESPT pathway or other pathway(s) responsible for LSS, the three-dimensional (3-D) structure of mAmetrine must be determined by performing X-ray crystallography. X-ray crystallography involved a series of steps: cloning; expression; large-scale purification; crystallization; collection, integration, and refinement of diffraction data; and determination of atomic positions. The protein crystallization technique used was hanging drop vapor-diffusion technique. The 3-D structure of mAmetrine was determined at resolution 2.1(A|°)and the data was analyzed to study the mechanism of ESPT pathway or other pathway(s) responsible for LSS. Further site-directed mutagenesis (F203Y, S205N and E222K) were required to finalize the structural analysis of mAmetrine.