Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction.

Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes' innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity. Here, we investigate the structural features that control the stereoselectivity in an oxidative dearomatization reaction that is key to making azaphilone natural products. Crystal structures of enantiocomplementary biocatalysts guided the development of multiple hypotheses centered on the structural features that control the stereochemical outcome of the reaction; however, in many cases, direct substitutions of active site residues in natural proteins led to inactive enzymes. Ancestral sequence reconstruction (ASR) and resurrection were employed as an alternative strategy to probe the impact of each residue on the stereochemical outcome of the dearomatization reaction. These studies suggest that two mechanisms are active in controlling the stereochemical outcome of the oxidative dearomatization reaction: one involving multiple active site residues in AzaH and the other dominated by a single Phe to Tyr switch in TropB and AfoD. Moreover, this study suggests that the flavin-dependent monooxygenases (FDMOs) adopt simple and flexible strategies to control stereoselectivity, which has led to stereocomplementary azaphilone natural products produced by fungi. This paradigm of combining ASR and resurrection with mutational and computational studies showcases sets of tools for understanding enzyme mechanisms and provides a solid foundation for future protein engineering efforts.

Guiding discovery of protein sequence-structure-function modeling.

MOTIVATION: Protein engineering techniques are key in designing novel catalysts for a wide range of reactions. Although approaches vary in their exploration of the sequence-structure-function paradigm, they are often hampered by the labor-intensive steps of protein expression and screening. In this work, we describe the development and testing of a high-throughput in silico sequence-structure-function pipeline using AlphaFold2 and fast Fourier transform docking that is benchmarked with enantioselectivity and reactivity predictions for an ancestral sequence library of fungal flavin-dependent monooxygenases. RESULTS: The predicted enantioselectivities and reactivities correlate well with previously described screens of an experimentally available subset of these proteins and capture known changes in enantioselectivity across the phylogenetic tree representing ancestorial proteins from this family. With this pipeline established as our functional screen, we apply ensemble decision tree models and explainable AI techniques to build sequence-function models and extract critical residues within the binding site and the second-sphere residues around this site. We demonstrate that the top-identified key residues in the control of enantioselectivity and reactivity correspond to experimentally verified residues. The in silico sequence-to-function pipeline serves as an accelerated framework to inform protein engineering efforts from vast informative sequence landscapes contained in protein families, ancestral resurrects, and directed evolution campaigns. AVAILABILITY: Jupyter notebooks detailing the sequence-structure-function pipeline are available at https://github.com/BrooksResearchGroup-UM/seq_struct_func.

Exploring the Limits of the Generalized CHARMM and AMBER Force Fields through Predictions of Hydration Free Energy of Small Molecules.

Accurate force field parameters, potential energy functions, and receptor-ligand models are essential for modeling the solvation and binding of drug-like molecules to a receptor. A large and ever-growing chemical space of medicinally relevant scaffolds has also required these factors, especially force field parameters, to be highly transferable. Generalized force fields such as the CHARMM General Force Field (CGenFF) and the generalized AMBER force field (GAFF) have accomplished this feat along with other contemporaneous ones like OPLS. Here, we analyze the limits in the parametrization of drug-like small molecules by CGenFF and GAFF in terms of the various functional groups represented within them. Specifically, we link the presence of specific functional groups to the error in the absolute hydration free energy of over 600 small molecules, predicted by alchemical free energy methods implemented in the CHARMM program. Our investigation reveals that molecules with (i) a nitro group in CGenFF and GAFF are, respectively, over- or undersolubilized in aqueous medium, (ii) amine groups are undersolubilized more so in CGenFF than in GAFF, and (iii) carboxyl groups are more oversolubilized in GAFF than in CGenFF. We present our analyses of the potential factors underlying these trends. We also showcase the use of a machine-learning-based approach combined with the SHapley Additive exPlanations framework to attribute these trends to specific functional groups, which can be easily adopted to explore the limits of other general force fields.

Fabrication of an Efficient Planar Organic-Silicon Hybrid Solar Cell with a 150 nm Thick Film of PEDOT: PSS.

Organic-inorganic hybrid solar cells composed of p-type conducting polymer poly (3,4-ethylene-dioxythiophene): polystyrenesulfonate (PEDOT: PSS) and n-type silicon (Si) have gained considerable interest in recent years. From this viewpoint, we present an efficient hybrid solar cell based on PEDOT: PSS and the planar Si substrate (1 0 0) with the simplest and cost-effective experimental procedures. We study and optimize the thickness of the PEDOT: PSS film to improve the overall performance of the device. We also study the effect of ethylene glycol (EG) by employing a different wt % as a solvent in the PEDOT: PSS to improve the device's performance. Silver (Ag) was deposited by electron beam evaporation as the front and rear contacts for the solar cell device. The whole fabrication process was completed in less than three hours. A power conversion efficiency (PCE) of 5.1%, an open circuit voltage (Voc) of 598 mV, and a fill factor (FF) of 58% were achieved.

In situ relative self-dependent calibration of electron cyclotron emission imaging via shape matching.

Electron Cyclotron Emission Imaging (ECEI) is a diagnostic system which measures 2-D electron temperature profiles with high spatial-temporal resolution. Usually only the normalized electron temperature fluctuations are utilized to investigate the magnetohydrodynamics modes due to the difficulties of ECEI calibration. In this paper, we developed a self-dependent calibration method for 24 × 16 channel high-resolution ECEI on the Experimental Advanced Superconducting Tokamak. The technique of shape matching is applied to solve for the matrix of the calibration coefficients. The calibrated area is further expanded to an occupation ratio of 88% observation area by utilizing the features of sawtooth crash. The result is self-consistent and consistent with calibrated 1D ECE measurement.