Crystal Structure of Venus, a Yellow Fluorescent Protein with Improved Maturation and Reduced Environmental Sensitivity

Agata Rekas, Jean-René Alattia, Takeharu Nagai, Atsushi Miyawaki, Mitsuhiko Ikura
2002 Journal of Biological Chemistry  
Yellow emission variants of green fluorescent protein (GFP) have been found useful in a variety of applications in biological systems due to their red-shifted emission spectrum and sensitivity to environmental parameters, such as pH and ionic strength. However, slow maturation properties and new requirements for more intense fluorescence necessitated further mutagenesis studies of these proteins. Venus, a new variant with improved maturation and brightness, as well as reduced environmental
more » ... environmental dependence, was recently developed by introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP). In this paper, we present the crystal structure of Venus at 2.2 Å resolution, which enabled us to correlate its novel features with these mutation points. The rearrangement of several side chains near the chromophore, initiated by the F46L mutation, was found to improve maturation at 37°C by removing steric and energetic constraints, which may hinder folding of the polypeptide chain, and by accelerating the oxidation of the C␣-C␤ bond of Tyr 66 during chromophore formation. M153T, V163A, and S175G were also found to improve the rate of maturation by creating regions of greater flexibility. F64L induced large conformational changes in the molecule, leading to the removal of halide sensitivity by preventing ion access to the binding site. Green fluorescent protein (GFP), 1 originally isolated from the jellyfish Aequorea victoria, has been the subject of continued interest since its gene was first cloned in 1992 (1). The high stability of mature GFP over various environment conditions, the spontaneous autocatalytical generation of the fluorophore of GFP, and the possibility of spectral manipulation by mutagenesis, make GFP and its related proteins attractive tools for numerous biological applications (2). Maturation of GFP proceeds in three major steps, beginning
doi:10.1074/jbc.m209524200 pmid:12370172 fatcat:ah5gznkh25d5hjeqoto72wecam