Rational Design of a Circularly Permuted Flavin-Based Fluorescent Protein.

Flavin-based fluorescent proteins are oxygen-independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering.

Similar enzymatic functions in distinct bioluminescence systems: evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Beyond the Green Fluorescent Protein: Biomolecular Reporters for Anaerobic and Deep-Tissue Imaging.

Fluorescence imaging represents cornerstone technology for studying biological function at the cellular and molecular levels. The technology's centerpiece is a prolific collection of genetic reporters based on the green fluorescent protein (GFP) and related analogs. More than two decades of protein engineering have endowed the GFP repertoire with an incredible assortment of fluorescent proteins, allowing scientists immense latitude in choosing reporters tailored to various cellular and environmental contexts. Nevertheless, GFP and derivative reporters have specific limitations that hinder their unrestricted use for molecular imaging. These challenges have inspired the development of new reporter proteins and imaging mechanisms. Here, we review how these developments are expanding the frontiers of reporter gene techniques to enable nondestructive studies of cell function in anaerobic environments and deep inside intact animals-two important biological contexts that are fundamentally incompatible with the use of GFP-based reporters.

Similar enzymatic functions in distinct bioluminescence systems: Evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii , transfer sulfate in vivo to the luciferin substrate, vargulin. We find luciferin sulfotransferases of ostracods are not orthologous to known luciferin sulfotransferases of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Re-use of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non-productive binding: insights from crystal structures and MD simulation.

Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p-nitrophenyl-ß-d-cellobioside (pNPC), p-nitrophenyl-ß-d-lactoside (pNPL) and methylumbelliferyl-ß-d-cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o-nitrophenyl-ß-d-cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non-productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non-productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (kcat ) than TrCel7A, but higher Michaelis-Menten (KM ) constants as well. Furthermore, oNPC turned out to be useful as an active-site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.

Characterization of flavin binding in oxygen-independent fluorescent reporters.

Fluorescent proteins based on light, oxygen, and voltage (LOV) sensing photoreceptors are among the few reporter gene technologies available for studying living systems in oxygen-free environments that render reporters based on the green fluorescent protein nonfluorescent. LOV reporters develop fluorescence by binding flavin mononucleotide (FMN), which they endogenously obtain from cells. As FMN is essential to cell physiology as well as for determining fluorescence in LOV proteins, it is important to be able to study and characterize flavin binding in LOV reporters. To this end, we report a method for reversibly separating FMN from two commonly used LOV reporters to prepare stable and soluble apoproteins. Using fluorescence titration, we measured the equilibrium dissociation constant for binding with all three cellular flavins: FMN, flavin adenine dinucleotide, and riboflavin. Finally, we exploit the riboflavin affinity of apo LOV reporters, identified in this work, to develop a fluorescence turn-on biosensor for vitamin B2.

Plant α-glucan phosphatases SEX4 and LSF2 display different affinity for amylopectin and amylose.

The plant glucan phosphatases Starch EXcess 4 (SEX4) and Like Sex Four2 (LSF2) apply different starch binding mechanisms. SEX4 contains a carbohydrate binding module, and LSF2 has two surface binding sites (SBSs). We determined KDapp for amylopectin and amylose, and KD for ß-cyclodextrin and validated binding site mutants deploying affinity gel electrophoresis (AGE) and surface plasmon resonance. SEX4 has a higher affinity for amylopectin; LSF2 prefers amylose and ß-cyclodextrin. SEX4 has 50-fold lower KDapp for amylopectin compared to LSF2. Molecular dynamics simulations and AGE data both support long-distance mutual effects of binding at SBSs and the active site in LSF2.