Structural Characterization of a Blue Chromoprotein and Its Yellow Mutant from the Sea Anemone Cnidopus Japonicus

Abstract

Green fluorescent protein (GFP) and its relatives (GFP protein family) have been isolated from marine organisms such as jellyfish and corals that belong to the phylum Cnidaria (stinging aquatic invertebrates). They are intrinsically fluorescent proteins. In search of new members of the family of green fluorescent protein family, we identified a non-fluorescent chromoprotein from the Cnidopus japonicus species of sea anemone that possesses 45% sequence identity to dsRed (a red fluorescent protein). This newly identified blue color protein has an absorbance maximum of 610 nm and is hereafter referred to as cjBlue. Determination of the cjBlue 1. 64 with Leu in wild-type cjBlue produced a visible color change from blue to yellow with a new absorbance maximum of 417 nm. Interestingly, the crystal structure of the yellow mutant Y64L revealed two His 197 imidazole ring orientations, suggesting a flip-flop interconversion between the two conformations in solution. We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins.The green fluorescent protein (GFP) 4 from Aequorea victoria has gained widespread interest as a biological reporter in living cells (1). Since its discovery, considerable efforts have been devoted to protein engineering, in conjunction with isolation of new GFP homologs, to expand the visible spectrum and properties of GFP protein family (1, 2). Characterized GFP protein family can be divided into two groups, the fluorescent proteins (FPs) and the non-fluorescent chromoproteins (CPs) (3, 4). The GFP chromophore arises through a unique autocatalytic posttranslational modification of a tripeptide, usually X-Tyr-Gly, in the primary sequence. The conformation and interaction of the chromophore with its local environment determines the spectral properties of the protein. X-ray crystallographic studies (5-7) have revealed the general relationship between the trans non-co-planarity of chromophores found in CPs and the cis co-planarity of chromophores found in FPs, with the exception of eqFP611, which has a trans co-planar chromophore.To date, four CPs from the Anthozoan species have been characterized: Rtms5 from Montipora efflorescens (8), gtCP from Goniopora tenuidens (9), aeCP597 from Actinia equine (10), and asFP595 from Anemonia sulcata (KFP) (5). Threedimensional structures of Rtms5 (6) and KFP (5) have been solved previously, both of which show the same fold as GFP and contain an internal chromophore. Studied CPs have exhibited absorbance maxima in a confined range of 560 -597 nm (11-14); no CP has been thus far found to absorb at absorbance maxima greater than 600 nm.Here we present a new CP from the Cnidopus japonicus sea anemone species, which absorbs at 610 nm. We report the molecular cloning, characterization and structure determination of this blue CP, hereafter termed cjBlue. We have also generated a yellow mutant variant from cjBlue with a single mutation at the 64 position (Tyr to Leu) using semi-random mutagenesis....

Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states

“…The data indicated that Tyr-67 does not form the C ␣ -C ␤ double bond. Interestingly, a spectrally similar chromophore, possibly also consisting of a single imidazolone ring with an N-acylimine bond, has been observed in a mutant of the cjBlue fluorescent protein with the Tyr67Leu substitution in the chromophore-forming tripeptide (24).…”

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.…”

Single-molecule spectroscopy of fluorescent proteins

“…The first protein of this group was DsRed [25], followed by many more discovered in other organisms [25][26][27][28][29][30][31][32] and by genetic modification of known proteins [33][34][35][36][37][38]. Remarkably, in all these proteins the structural motif of a barrel formed by β-sheets is conserved, as is the autocatalytic formation of the chromophore which is encapsulated within the protein barrel.…”

“…The discovery 1 [14], subsequent cloning [15], and application of the Aequoria green fluorescent protein [16], and the development of new colours of fluorescent proteins by protein engineering of Aequoria GFP [17][18][19][20][21][22][23][24], has provided revolutionary new capabilities to visualize molecular and cellular biological processes. The palette of fluorescent proteins has been enormously extended by the discovery of new intrinsically fluorescent visible fluorescent proteins (VFPs) from other marine organisms [25][26][27][28][29][30][31][32], and their optimization [33][34][35][36][37][38]. The combination of these genetically encodable markers with advanced microscopic and spectroscopic techniques has enabled quantitative measurement of protein-protein interactions.…”