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 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 as the 238-residue single GFP polypeptide folds into its nearly native conformation. Residues 65-67 of the folded protein then undergo several chemical reactions necessary for chromophore formation, including cyclization and dehydration (3). The final rate-limiting step in the maturation process involves the oxidation of the C␣-C␤ bond of Tyr 66 by aerial oxygen. This maturation process takes ϳ3 h at room temperature and its efficiency further decreases at 37°C, hindering the use of GFP in some biological applications. To improve maturation and performance at 37°C, mutations such as F99S, M153T, and V163A have been introduced, creating valuable GFP variants for a wider range of applications (4, 5).Thorough understanding of the relationship between the protein sequence and its physico-chemical properties requires three-dimensional structure determination of GFP at atomic resolution. Crystal structures of both the monomeric and dimeric forms of GFP have been solved previously (6, 7). The wild-type GFP (wtGFP) chromophore, which exists in equilibrium between two ground states, produces two maxima in the light absorption spectrum. Th...

Saposins Aa nd Ca re sphingolipid activator proteins requiredf or thel ysosomal breakdowno f galactosylceramide and glucosylceramide,r espectively.T he saposins interact with lipids, leading to an enhanced accessibility of thel ipid headgroups to their cognate hydrolases. We have determinedt he crystal structures of humans aposinsAandCt o2 .0 A˚and 2.4Å ,r espectively,a nd both reveal the compact, monomeric saposin fold. We confirmed that these twoproteins were monomeric in solution at pH 7.0b yanalytical centrifugation. However, at pH 4.8,inthe presence of thedetergent C 8 E 5 ,saposin Aa ssembled intod imers, while saposin Cf ormed trimers. Saposin Bw as dimeric under allc onditions tested.T he self-associationo ft he saposins is likelyt ob er elevantt oh ow theses mall proteins interact with lipids, membranes, andh ydrolase enzymes.Keywords: saposins;X -ray crystallography; analyticalu ltracentrifugation; protein-detergent interactions Saposins A, B, C, and Da re small, nonenzymatic proteins required for the breakdown of glycosphingolipids within the lysosome (Kolter and Sandhoff2005). They are derived from the proteolytic processing of the precursor protein prosaposin, producing the four individual saposins. The four saposin domains contained within prosaposin most likely arose from two tandem duplications of an ancestral gene into one single copy gene (Hazkani-Covoetal. 2002). Each saposin ''activates''the breakdown of particular lipid substrates by facilitating the access of the lipid headgroups to the active sites of cognate hydrolases. It is generally believed that in the absence of sphingolipid activator proteins, the oligosaccharide chains of the membranebound lipids do not extend far enough into the lysosomal lumen to be accessible to the active sites of the hydrolases. Mechanistically,t he saposins appear to activate lipid hydrolysis by solubilizing the lipid substrates or possibly by destabilizing the membrane structure (Vaccaro et al. 1993(Vaccaro et al. , 1995(Vaccaro et al. , 1997Wilkening et al. 1998;Salvioli et al. 2000). Article publishedonline aheado fp rint.A rticle and publicationd ate area th ttp://www.proteinscience.org/cgi

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Acid ␤-glucosidase (GCase) is a soluble lysosomal enzyme responsible for the hydrolysis of glucose from glucosylceramide and requires activation by the small nonenzymatic protein saposin C (sapC) to gain access to the membrane-embedded glycosphingolipid substrate. We have used in situ atomic force microscopy (AFM) with simultaneous confocal and epifluorescence microscopies to investigate the interactions of GCase and sapC with lipid bilayers. GCase binds to sites on membranes transformed by sapC, and enzyme activity occurs at loci containing both GCase and sapC. Using FRET, we establish the presence of GCase/sapC and GCase/ product contacts in the bilayer. These data support a mechanism in which sapC locally alters regions of bilayer for subsequent attack by the enzyme in stably bound protein complexes.atomic force microscopy ͉ confocal microscopy ͉ FRET ͉ interfacial catalysis ͉ lipid storage disease