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....
Lipopeptide detergents (LPDs) are a new class of amphiphile designed specifically for the structural study of integral membrane proteins. The LPD monomer consists of a 25-residue peptide with fatty acyl chains linked to side chains located at positions 2 and 24 of the peptide. LPDs are designed to form ␣-helices that selfassemble into cylindrical micelles, providing a more natural interior acyl chain packing environment relative to traditional detergents. We have determined the crystal structure of LPD-12, an LPD coupled to two dodecanoic acids, to a resolution of 1.20 Å. The LPD-12 monomers adopt the target conformation and associate into cylindrical octamers as expected. Pairs of helices are strongly associated as Alacoil-type antiparallel dimers, and four of these dimers interact through much looser contacts into assemblies with approximate D 2 symmetry. The aligned helices form a cylindrical shell with a hydrophilic exterior that protects an interior hydrophobic cavity containing the 16 LPD acyl chains. Over 90% of the methylene/methyl groups from the acylated side chains are visible in the micelle interiors, and Ϸ90% of these adopt trans dihedral angle conformations. Dodecylmaltoside (DDM) was required for the crystallization of LPD-12, and we find 10 -24 ordered DDM molecules associated with each LPD assembly, resulting in an overall micelle molecular weight of Ϸ30 kDa. The structures confirm the major design objectives of the LPD framework, and reveal unexpected features that will be helpful in the engineering additional versions of lipopeptide amphiphiles.de novo protein design ͉ detergent design ͉ membrane proteins ͉ self-assembling amphiphiles ͉ x-ray crystallography D etergents that are able to stabilize native structures of membrane proteins in the absence of lipid bilayers are essential tools for the structural study of membrane proteins (1, 2). Despite the large number of detergents that are available, no single detergent is optimal for all purposes, and choice of the solubilizing agent is highly dependent on both the target protein and on the application. In particular, the structural study of membrane proteins by NMR or x-ray crystallography depends critically on the choice of detergent (3, 4), and many proteins cannot be studied because of problems with protein stability and aggregation in the commonly used detergents (5). Because of these issues, there is a need to expand the range of amphiphiles available for membrane proteins research.Nondenaturing detergents act as membrane mimetics, and an ideal detergent would generate a local environment at the protein surface that is indistinguishable from that of the native lipid bilayer. A central shortcoming of many of the commonly used detergents is that the micelle interior is less ordered and less well-packed relative to the interiors of lipid bilayers (2, 6). This is a direct result of the shape of the amphiphile monomer, which has a high degree of positive intrinsic curvature, resulting in self-assembly into spherical or ellipsoidal micelles with...
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