Transmembrane Shuttling of Photosynthetically Produced Electrons to Propel Extracellular Biocatalytic Redox Reactions in a Modular Fashion.

Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time-consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme- or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.

Mapping the transition state for a binding reaction between ancient intrinsically disordered proteins.

Intrinsically disordered protein domains often have multiple binding partners. It is plausible that the strength of pairing with specific partners evolves from an initial low affinity to a higher affinity. However, little is known about the molecular changes in the binding mechanism that would facilitate such a transition. We previously showed that the interaction between two intrinsically disordered domains, NCBD and CID, likely emerged in an ancestral deuterostome organism as a low-affinity interaction that subsequently evolved into a higher-affinity interaction before the radiation of modern vertebrate groups. Here we map native contacts in the transition states of the low-affinity ancestral and high-affinity human NCBD/CID interactions. We show that the coupled binding and folding mechanism is overall similar but with a higher degree of native hydrophobic contact formation in the transition state of the ancestral complex and more heterogeneous transient interactions, including electrostatic pairings, and an increased disorder for the human complex. Adaptation to new binding partners may be facilitated by this ability to exploit multiple alternative transient interactions while retaining the overall binding and folding pathway.

Boronate affinity electrophoresis for the purification and analysis of cofactor-modified RNAs.

RNA modifications are widely distributed in Nature, and their thorough analysis helps answering fundamental biological questions. Nowadays, mass spectrometry or deep-sequencing methods are often used for the analysis. With the raising number of newly discovered RNA modifications, such as the 5'-NAD cap in Escherichia coli, there is an important need for new, less complex and fast analytical tools to analyze the occurrence, amount, and distribution of modified RNAs in cells. To accomplish this task, we have revisited the previously developed affinity gel electrophoresis principles and copolymerized acryloylaminophenyl boronic acid (APB) in standard denaturing polyacrylamide gels to retard the NAD- or FAD-modified RNAs compared to the unmodified RNAs in the gels. The boronyl groups inside the gel form relatively stable complexes with 1,2-cis diols, occurring naturally at the 3'-end of RNA, and also in the nicotinamide riboside of NAD-modified RNA at the 5'-end. The transient formation of diesters between the immobilized boronic acid and the diols causes lower mobility of the modified RNAs, compared to unmodified RNAs, resulting in two distinct bands for one RNA sequence. We used APB affinity gel electrophoresis to preparatively purify in vitro transcribed NAD-RNA from triphosphorylated RNA, to study the enzyme kinetics of the NAD-RNA decapping enzyme NudC, and to determine the NAD modification ratios of various cellular sRNAs. In summary, APB affinity gels can be used to study cofactor-modified RNAs with low amounts of material, and to rapidly screen for their occurrence in total RNA while avoiding complex sample treatments.

Solvent concentration at 50% protein unfolding may reform enzyme stability ranking and process window identification.

As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is required which displays high stability in the presence of this co-solvent. Consequently, it is of utmost importance to identify the most suitable enzyme or the appropriate reaction conditions. Until now, the melting temperature is used in general as a measure for stability of enzymes. The experiments here show, that the melting temperature does not correlate to the activity observed in the presence of the solvent. As an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T in short c U 50 T is introduced. Analyzing a set of ene reductases, c U 50 T is shown to indicate the concentration of the co-solvent where also the activity of the enzyme drops fastest. Comparing possible rankings of enzymes according to melting temperature and c U 50 T reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of c U 50 versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature.

The dynamic properties of a nuclear coactivator binding domain are evolutionarily conserved.

Evolution of proteins is constrained by their structure and function. While there is a consensus that the plasticity of intrinsically disordered proteins relaxes the structural constraints on evolution there is a paucity of data on the molecular details of these processes. The Nuclear Coactivator Binding Domain (NCBD) from CREB-binding protein is a protein interaction domain, which contains a hydrophobic core but is not behaving as a typical globular domain, and has been described as 'molten-globule like'. The highly dynamic properties of NCBD makes it an interesting model system for evolutionary structure-function investigation of intrinsically disordered proteins. We have here compared the structure and biophysical properties of an ancient version of NCBD present in a bilaterian animal ancestor living around 600 million years ago with extant human NCBD. Using a combination of NMR spectroscopy, circular dichroism and kinetics we show that although NCBD has increased its thermodynamic stability, it has retained its dynamic biophysical properties in the ligand-free state in the evolutionary lineage leading from the last common bilaterian ancestor to humans. Our findings suggest that the dynamic properties of NCBD have been maintained by purifying selection and thus are important for its function, which includes mediating several distinct protein-protein interactions.

Transmembrane Shuttling of Photosynthetically Produced Electrons to Propel Extracellular Biocatalytic Redox Reactions in a Modular Fashion.

Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time-consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme- or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.

PQQ-dependent Dehydrogenase Enables One-pot Bi-enzymatic Enantio-convergent Biocatalytic Amination of Racemic sec-Allylic Alcohols.

The asymmetric amination of secondary racemic allylic alcohols bears several challenges like the reactivity of the bi-functional substrate/product as well as of the α,ß-unsaturated ketone intermediate in an oxidation-reductive amination sequence. Heading for a biocatalytic amination cascade with a minimal number of enzymes, an oxidation step was implemented relying on a single PQQ-dependent dehydrogenase with low enantioselectivity. This enzyme allowed the oxidation of both enantiomers at the expense of iron(III) as oxidant. The stereoselective amination of the α,ß-unsaturated ketone intermediate was achieved with transaminases using 1-phenylethylamine as formal reducing agent as well as nitrogen source. Choosing an appropriate transaminase, either the (R)- or (S)-enantiomer was obtained in optically pure form (>98 % ee). The enantio-convergent amination of the racemic allylic alcohols to one single allylic amine enantiomer was achieved in one pot in a sequential cascade.

Kinetic and structural roles for the surface in guiding SAS-6 self-assembly to direct centriole architecture.

Discovering mechanisms governing organelle assembly is a fundamental pursuit in biology. The centriole is an evolutionarily conserved organelle with a signature 9-fold symmetrical chiral arrangement of microtubules imparted onto the cilium it templates. The first structure in nascent centrioles is a cartwheel, which comprises stacked 9-fold symmetrical SAS-6 ring polymers emerging orthogonal to a surface surrounding each resident centriole. The mechanisms through which SAS-6 polymerization ensures centriole organelle architecture remain elusive. We deploy photothermally-actuated off-resonance tapping high-speed atomic force microscopy to decipher surface SAS-6 self-assembly mechanisms. We show that the surface shifts the reaction equilibrium by ~104 compared to solution. Moreover, coarse-grained molecular dynamics and atomic force microscopy reveal that the surface converts the inherent helical propensity of SAS-6 polymers into 9-fold rings with residual asymmetry, which may guide ring stacking and impart chiral features to centrioles and cilia. Overall, our work reveals fundamental design principles governing centriole assembly.

Kinome-Wide Profiling Prediction of Small Molecules.

Extensive kinase profiling data, covering more than half of the human kinome, are available nowadays and allow the construction of activity prediction models of high practical utility. Proteochemometric (PCM) approaches use compound and protein descriptors, which enables the extrapolation of bioactivity values to thus far unexplored kinases. In this study, the potential of PCM to make large-scale predictions on the entire kinome is explored, considering the applicability on novel compounds and kinases, including clinically relevant mutants. A rigorous validation indicates high predictive power on left-out kinases and superiority over individual kinase QSAR models for new compounds. Furthermore, external validation on clinically relevant mutant kinases reveals an excellent predictive power for mutations spread across the ATP binding site.

Folding Optimization In Vivo Uncovers New Chaperones.

By employing a genetic selection that forces the cell to fold an unstable, aggregation-prone test protein in order to survive, we have generated bacterial strains with enhanced periplasmic folding capacity. These strains enhance the soluble steady-state level of the test protein. Most of the bacterial variants we isolated were found to overexpress one or more periplasmic proteins including OsmY, Ivy, DppA, OppA, and HdeB. Of these proteins, only HdeB has convincingly been previously shown to function as chaperone in vivo. By giving bacteria the stark choice between death and stabilizing a poorly folded protein, we have now generated designer bacteria selected for their ability to stabilize specific proteins.