Characterizing protein protonation microstates using Monte Carlo sampling

Proteins are polyelectrolytes with acidic or basic amino acids making up ≈25% of the residues. The protonation state of all Asp, Glu, Arg, Lys, His and other protonatable residues, cofactors and ligands define each protonation microstate. As all of these residues will not be fully ionized or neutral, proteins exist in a mixture of microstates. The microstate distribution changes with pH. As the protein environment modifies the proton affinity of each site the distribution may also change in different reaction intermediates or as ligands are bound. Particular protonation microstates may be required for function, while others exist simply because there are many states with similar energy. Here, the protonation microstates generated in Monte Carlo sampling in MCCE are characterized in HEW lysozyme as a function of pH and bacterial photosynthetic reaction centers (RCs) in different reaction intermediates. The lowest energy and highest probability microstates are compared. The ∆G, ∆H and ∆S between the four protonation states of Glu35 and Asp52 in lysozyme are shown to be calculated with reasonable precision. A weighted Pearson correlation analysis identifies coupling between residue protonation states in RCs and how they change when the quinone in the QB site is reduced.

Analysis of Fluctuation in the Heme-Binding Pocket and Heme Distortion in Hemoglobin and Myoglobin

Heme is located in the active site of proteins and has diverse and important biological functions, such as electron transfer and oxygen transport and/or storage. The distortion of heme porphyrin is considered an important factor for the diverse functions of heme because it correlates with the physical properties of heme, such as oxygen affinity and redox potential. Therefore, clarification of the relationship between heme distortion and the protein environment is crucial in protein science. Here, we analyzed the fluctuation in heme distortion in the protein environment for hemoglobin and myoglobin using molecular dynamics (MD) simulations and quantum mechanical (QM) calculations. We also investigated the protein structures of hemoglobin and myoglobin stored in Protein Data Bank and found that heme is distorted along the doming mode, which correlates with its oxygen affinity, more prominently in the protein environment than in the isolated state, and the magnitude of distortion is different between hemoglobin and myoglobin. This tendency was also observed in the results of MD simulations and QM calculations. These results suggest that heme distortion is affected by its protein environment and fluctuates around its fitted conformation, leading to physical properties that are appropriate for protein functions.

Influence of the Greater Protein Environment on the Electrostatic Potential in Metalloenzyme Active Sites: the Case of Formate Dehydrogenase

The Mo/W containing metalloenzyme formate dehydrogenase (FDH) is an efficient and selective natural catalyst which reversibly converts CO2 to formate under ambient conditions. A greater understanding of the role of the protein environment in determining the local properties of the FDH active site would enable rational bioinspired catalyst design. In this study, we investigate the impact of the greater protein environment on the electrostatic potential (ESP) of the active site. To model the enzyme environment, we used a combination of long-timescale classical molecular dynamics (MD) and multiscale quantum-mechanical/molecular-mechanical (QM/MM) simulations. We leverage the charge shift analysis method to systematically construct QM regions and analyze the electronic environment of the active site by evaluating the degree of charge transfer between the core active site and the protein environment. The contribution of the terminal chalcogen ligand to the ESP of the metal center is substantial and dependent on the chalcogen identity, with ESPs less negative and similar for Se and S terminal chalcogens than for O regardless of whether the Mo6+ or W6+ metal center is present. Our evaluation reveals that the orientation of the sidechains and ligand conformations will alter the relative trends in the ESP observed for a given metal center or terminal chalcogen, highlighting the importance of sampling dynamic fluctuations in the protein. Overall, our observations suggest that the terminal chalcogen ligand identity plays an important role in the enzymatic activity of FDH.

Antibacterial, Antifungal Chalcone Derivatives as EGFR Inhibitors-Molecular Docking and ADMET Studies

Aim: In our earlier research, we have synthesized series of substituted 1-(2, 5-dimethyl thiophene-3yl)-(4-substituted phenyl)-2-propene-1-one derivatives and evaluated them for their anti-bacterial and antifungal activity. In recent years, chalcone derivatives are proved for their varied pharmacological effects ranging from antimicrobial activity to anti-cancer effects. In this study, we have hypothesized the efficiency of our earlier synthesized anti-bacterial and antifungal chalcone derivatives for their potential inhibition of epidermal growth factor receptor protein (EGFR), through molecular docking studies. Methodology: Molecular docking simulation studies are performed using the Glide XP module of Schrodinger Suite and ligand binding energies are also calculated. Results: Molecular docking studies of the selected compounds against EGFR revealed docking scores ranging from -6.746 (compound 5) to -5.681 (compound 3) and also provided insight into binding conformations of the ligands in the EGFR protein environment. Additionally, molecular property and Absorption, Distribution, Metabolism, and Excretion (ADME) predictor analysis is also performed for the dataset ligands, which further provided the probable explanation for the binding potentials. Conclusion: Among all the tested dataset ligands, compound 5 has shown the highest dock score (-6.746) with better ADME profiles. Binding energies in the protein-ligand interactions explain how fit the ligand binds with the target protein. Molecular docking studies of these anti-bacterial, antifungal chalcone derivatives provided deeper insights in understanding the probable conformations of these tested ligands in the EGFR protein environment.

Targeted Activation in Localized Protein Environments via Deep Red Photoredox Catalysis

State-of-the art photoactivation strategies in chemical biology provide spatiotemporal control and visualization of biological processes. However, using high energy light (λ < 500 nm) for substrate or photocatalyst sensitization can lead to background activation of photoactive small molecule probes and reduce its efficacy in complex biological environments. Here we describe the development of targeted aryl azide activation via deep red light (λ = 660 nm) photoredox catalysis and its use in photocatalyzed proximity labeling. We demonstrate that aryl azides are converted to triplet nitrenes via a novel redox-centric mechanism and show that its spatially localized-formation requires both red light and a photocatalyst-targeting modality. This technology was applied in different colon cancer cell systems for targeted protein environment labeling of epithelial cell adhesion molecule (EpCAM). We identified a small subset of proteins with previously known and unknown association to EpCAM, including CDH3, a clinically relevant protein that shares high tumor selective expression with EpCAM.

Electronic Couplings and Electrostatic Interactions Behind the Light Absorption of Retinal Proteins

The photo-functional chromophore retinal exhibits a wide variety of optical absorption properties depending on its intermolecular interactions with surrounding proteins and other chromophores. By utilizing these properties, microbial and animal rhodopsins express biological functions such as ion-transport and signal transduction. In this review, we present the molecular mechanisms underlying light absorption in rhodopsins, as revealed by quantum chemical calculations. Here, symmetry-adapted cluster-configuration interaction (SAC-CI), combined quantum mechanical and molecular mechanical (QM/MM), and transition-density-fragment interaction (TDFI) methods are used to describe the electronic structure of the retinal, the surrounding protein environment, and the electronic coupling between chromophores, respectively. These computational approaches provide successful reproductions of experimentally observed absorption and circular dichroism (CD) spectra, as well as insights into the mechanisms of unique optical properties in terms of chromophore-protein electrostatic interactions and chromophore-chromophore electronic couplings. On the basis of the molecular mechanisms revealed in these studies, we also discuss strategies for artificial design of the optical absorption properties of rhodopsins.

Proximity proteomics identifies PAK4 as a component of Afadin–Nectin junctions

Human PAK4 is an ubiquitously expressed p21-activated kinase which acts downstream of Cdc42. Since PAK4 is enriched in cell-cell junctions, we probed the local protein environment around the kinase with a view to understanding its location and substrates. We report that U2OS cells expressing PAK4-BirA-GFP identify a subset of 27 PAK4-proximal proteins that are primarily cell-cell junction components. Afadin/AF6 showed the highest relative biotin labelling and links to the nectin family of homophilic junctional proteins. Reciprocally >50% of the PAK4-proximal proteins were identified by Afadin BioID. Co-precipitation experiments failed to identify junctional proteins, emphasizing the advantage of the BioID method. Mechanistically PAK4 depended on Afadin for its junctional localization, which is similar to the situation in Drosophila. A highly ranked PAK4-proximal protein LZTS2 was immuno-localized with Afadin at cell-cell junctions. Though PAK4 and Cdc42 are junctional, BioID analysis did not yield conventional cadherins, indicating their spatial segregation. To identify cellular PAK4 substrates we then assessed rapid changes (12’) in phospho-proteome after treatment with two PAK inhibitors. Among the PAK4-proximal junctional proteins seventeen PAK4 sites were identified. We anticipate mammalian group II PAKs are selective for the Afadin/nectin sub-compartment, with a demonstrably distinct localization from tight and cadherin junctions.