Methods for the quantication of rhythmic biological signals have been essential for the discovery of function and design of biological oscillators. Advances in live measurements have allowed recordings of unprecedented resolution revealing a new world of complex heterogeneous oscillations with multiple noisy non-stationary features. However, our understanding of the underlying mechanisms regulating these oscillations has been lagging behind, partially due to the lack of simple tools to reliably quantify these complex non-stationary features. With this challenge in mind, we have developed pyBOAT, a Python-based fully automatic stand-alone software that integrates multiple steps of non-stationary oscillatory time series analysis into an easy-to-use graphical user interface. pyBOAT implements continuous wavelet analysis which is specically designed to reveal time-dependent features. In this work we illustrate the advantages of our tool by analyzing complex non-stationary time-series proles. Our approach integrates data-visualization, optimized sinc-lter detrending, amplitude envelope removal and a subsequent continuous-wavelet based timefrequency analysis. Finally, using analytical considerations and numerical simulations we discuss unexpected pitfalls in commonly used smoothing and detrending operations.
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.
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.
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.
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 to 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.
Discovering the physical principles directing organelle assembly is a fundamental pursuit in biology. Centrioles are evolutionarily conserved organelles with a 9-fold rotational symmetry of chiral microtubules imparted onto the cilia they template. Centriole assemble from likewise symmetrical ring polymers of SAS-6 proteins, orthogonal to a toroidal surface surrounding the resident centriole. How surface properties ensure ring assembly with proper symmetry and orthogonal arrangement is not known. Here, we deployed photothermally-actuated off-resonance tapping high-speed atomic force microscopy (PORT-HS-AFM) to decipher physical principles of surface-guided SAS-6 self-assembly. Using machine learning to quantify the polymerization reaction and developing a coagulation-fragmentation model, we discovered that the surface shifts the reaction equilibrium by ~104 compared to the solution situation, explaining orthogonal organelle emergence. Moreover, molecular dynamics and PORT-HS-AFM revealed that the surface converts helical SAS-6 polymers into 9-fold ring polymers with residual asymmetry, which may impart chiral features to centrioles and cilia. Overall, we discovered two fundamental physical principles directing robust centriole organelle assembly.
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,β‐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,β‐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.
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