The oxidation of the [4Fe-4S] cluster of DNA primase alters the binding energies with DNA and RNA primers.

DNA primase is an iron sulfur enzyme in DNA replication responsible for synthesizing short RNA primers that serve as starting points for DNA synthesis. The role of the [4Fe-4S] cluster is not well determined. Here, we calculate the redox potential of the [4Fe-4S] with and without DNA/RNA using continuum electrostatics. In addition, we identify the structural changes coupled to the oxidation/reduction. Our calculations show that the DNA/RNA primer lowers the redox potential by 110 and 50 mV for the [4Fe-4S]+ and [4Fe-4S]2+ states, respectively. The oxidation of the cluster is coupled to structural changes that significantly reduce the binding energies between the DNA and the nearby residues. The negative charges accumulated by the DNA and the RNA primers induce the oxidation of the [4Fe-4S] cluster. This in turn stimulates structural changes on the DNA-protein interface that significantly reduce the binding energies.

Mapping the Oxygens in the Oxygen-Evolving Complex of Photosystem II by Their Nucleophilicity Using Quantum Descriptors.

The oxygen-evolving complex (OEC) of Photosystem II catalyzes the water-splitting reaction using solar energy. Thus, understanding the reaction mechanism will inspire the design of biomimetic artificial catalysts that convert solar energy to chemical energy. Conceptual Density Functional Theory (CDFT) focuses on understanding the reactivity of molecules and the atomic contribution to the overall nucleophilicity and electrophilicity of the molecule using quantum descriptors. However, this method has not been applied to the OEC before. Here, we use Fukui functions and the dual descriptor to provide quantitative measures of the nucleophilicity and electrophilicity of oxygens in the OEC for different models in different S states. Our results show that the µ-oxo bridges connected to terminal Mn4 are nucleophilic, and those in the cube formed by Mn1, Mn2, and Mn3 are mostly electrophilic. The dual descriptors of the bridging oxygens in the OEC showed a similar reactivity to that of bridging oxygens in Mn model compounds. However, the terminal water W1, which is bound to Mn4, showed very strong reactivity in some of the S3 models. Thus, our calculations support the model that proposes the formation of the O2 molecule through nucleophilic attack by a terminal water.

Bridging the Coordination Chemistry of Small Compounds and Metalloproteins Using Machine Learning.

Metalloproteins require metal ions as cofactors to catalyze specific reactions with remarkable efficiency and specificity. In various electron transfer reactions, metals in the active sites change their oxidation states to facilitate the biochemical reactions. Cryogenic electron microscopy, X-ray, and X-ray free electron laser (XFEL) crystallography are used to image metalloproteins to understand the reaction mechanisms. However, radiation damage in cryoEM and X-ray crystallography, and the challenge of generating homogeneous crystals and keeping the appropriate experimental conditions for all the crystals in XFEL crystallography, may alter the oxidation states. Here, we build machine learning models trained on a large data set from the Cambridge Crystallographic Data Center to evaluate the metal oxidation states. The models yield high accuracy scores (from 82% to 94%) for all metals in the small molecules. Then, they were used to predict the oxidation states of more than 30 000 metal clusters in metalloproteins with Fe, Mn, Co, and Cu in their active sites. We found that most of the metals exist in the lower oxidation states (Fe2+ 77%, Mn2+ 85%, Co2+ 65%, and Cu+ 64%), and these populations correlate with the standard reduction potentials of the metal ions. Furthermore, we found no clear correlation between these populations and the resolution of the structures, which suggests no significant dependence of these predictions on the resolution. Our models represent a valuable tool for evaluating the oxidation states of the metals in metalloproteins imaged with different techniques. The data files and the machine learning code are available in a public GitHub repository: https://github.com/mamin03/OxitationStatesMetalloprotein.git.

Computing the Relative Affinity of Chlorophylls a and b to Light-Harvesting Complex II.

In plants and algae, the primary antenna protein bound to photosystem II is light-harvesting complex II (LHCII), a pigment-protein complex that binds eight chlorophyll (Chl) a molecules and six Chl b molecules. Chl a and Chl b differ only in that Chl a has a methyl group (-CH3) on one of its pyrrole rings, while Chl b has a formyl group (-CHO) at that position. This blue-shifts the Chl b absorbance relative to Chl a. It is not known how the protein selectively binds the right Chl type at each site. Knowing the selection criteria would allow the design of light-harvesting complexes that bind different Chl types, modifying an organism to utilize the light of different wavelengths. The difference in the binding affinity of Chl a and Chl b in pea and spinach LHCII was calculated using multiconformation continuum electrostatics and free energy perturbation. Both methods have identified some Chl sites where the bound Chl type (a or b) has a significantly higher affinity, especially when the protein provides a hydrogen bond for the Chl b formyl group. However, the Chl a sites often have little calculated preference for one Chl type, so they are predicted to bind a mixture of Chl a and b. The electron density of the spinach LHCII was reanalyzed, which, however, confirmed that there is negligible Chl b in the Chl a-binding sites. It is suggested that the protein chooses the correct Chl type during folding, segregating the preferred Chl to the correct binding site.

Ionizing radiation induces cross-linking of two noncovalently bound collagen mimetic peptide triple helices in the absence of a molecular environment.

Cross-linking is a fundamental molecular process that is highly important for many applications, in particular, to tune the properties of collagen-based biomaterials. Chemical reagents, the action of enzymes or physical factors such as heat or radiation can facilitate collagen cross-linking. Ionizing radiation has the advantages of being fast, efficient and free from potentially toxic reagents. Collagen cross-linking by ionizing radiation is thought to occur via a water-mediated pathway. In the past, synthesized collagen mimetic peptides have proven to be of great value for understanding the influence of the amino acid sequence on the stability of tertiary (fibrous) as well as secondary (triple helical) structures of collagen. Cross-linking of synthetic collagen mimetic peptides is often used for modifying the properties of biomaterials. In this work, for the first time, we apply radiation-induced cross-linking to synthetic collagen mimetic peptides and present an experimental and theoretical study of peptide hexamers consisting of two noncovalently bound triple helices in the absence of a molecular environment, i.e. in the gas phase. Our results show that X-ray photoabsorption of the hydroxylated hexamer leads to ionization and cross-linking of the two triple helices: thus, we found evidence that cross-linking can be achieved by ionizing radiation, without the presence of any reagent or water. We propose a cross-linking mechanism involving the creation of two radicals on hydroxyproline side-chains and their recombination, ultimately leading to covalent bond formation between the triple helices.

Structure of a dimeric photosystem II complex from a cyanobacterium acclimated to far-red light.

Photosystem II (PSII) is the water-splitting enzyme central to oxygenic photosynthesis. To drive water oxidation, light is harvested by accessory pigments, mostly chlorophyll (Chl) a molecules, which absorb visible light (400-700 nm). Some cyanobacteria facultatively acclimate to shaded environments by altering their photosynthetic machinery to additionally absorb far-red light (FRL, 700-800 nm), a process termed far-red light photoacclimation or FaRLiP. During far-red light photoacclimation, FRL-PSII is assembled with FRL-specific isoforms of the subunits PsbA, PsbB, PsbC, PsbD, and PsbH, and some Chl-binding sites contain Chls d or f instead of the usual Chl a. The structure of an apo-FRL-PSII monomer lacking the FRL-specific PsbH subunit has previously been determined, but visualization of the dimeric complex has remained elusive. Here, we report the cryo-EM structure of a dimeric FRL-PSII complex. The site assignments for Chls d and f are consistent with those assigned in the previous apo-FRL-PSII monomeric structure. All sites that bind Chl d or Chl f at high occupancy exhibit a FRL-specific interaction of the formyl moiety of the Chl d or Chl f with the protein environment, which in some cases involves a phenylalanine sidechain. The structure retains the FRL-specific PsbH2 subunit, which appears to alter the energetic landscape of FRL-PSII, redirecting energy transfer from the phycobiliprotein complex to a Chl f molecule bound by PsbB2 that acts as a bridge for energy transfer to the electron transfer chain. Collectively, these observations extend our previous understanding of the structure-function relationship that allows PSII to function using lower energy FRL.

Predicting the oxidation states of Mn ions in the oxygen-evolving complex of photosystem II using supervised and unsupervised machine learning.

Serial Femtosecond Crystallography at the X-ray Free Electron Laser (XFEL) sources enabled the imaging of the catalytic intermediates of the oxygen evolution reaction of Photosystem II (PSII). However, due to the incoherent transition of the S-states, the resolved structures are a convolution from different catalytic states. Here, we train Decision Tree Classifier and K-means clustering models on Mn compounds obtained from the Cambridge Crystallographic Database to predict the S-state of the X-ray, XFEL, and CryoEM structures by predicting the Mn's oxidation states in the oxygen-evolving complex. The model agrees mostly with the XFEL structures in the dark S1 state. However, significant discrepancies are observed for the excited XFEL states (S2, S3, and S0) and the dark states of the X-ray and CryoEM structures. Furthermore, there is a mismatch between the predicted S-states within the two monomers of the same dimer, mainly in the excited states. We validated our model against other metalloenzymes, the valence bond model and the Mn spin densities calculated using density functional theory for two of the mismatched predictions of PSII. The model suggests designing a more optimized sample delivery and illumiation systems are crucial to precisely resolve the geometry of the advanced S-states to overcome the noncoherent S-state transition. In addition, significant radiation damage is observed in X-ray and CryoEM structures, particularly at the dangler Mn center (Mn4). Our model represents a valuable tool for investigating the electronic structure of the catalytic metal cluster of PSII to understand the water splitting mechanism.

Computational Approach for Probing Redox Potential for Iron-Sulfur Clusters in Photosystem I.

Photosystem I is a light-driven electron transfer device. Available X-ray crystal structure from Thermosynechococcus elongatus showed that electron transfer pathways consist of two nearly symmetric branches of cofactors converging at the first iron-sulfur cluster FX, which is followed by two terminal iron-sulfur clusters FA and FB. Experiments have shown that FX has lower oxidation potential than FA and FB, which facilitates the electron transfer reaction. Here, we use density functional theory and Multi-Conformer Continuum Electrostatics to explain the differences in the midpoint Em potentials of the FX, FA and FB clusters. Our calculations show that FX has the lowest oxidation potential compared to FA and FB due to strong pairwise electrostatic interactions with surrounding residues. These interactions are shown to be dominated by the bridging sulfurs and cysteine ligands, which may be attributed to the shorter average bond distances between the oxidized Fe ion and ligating sulfurs for FX compared to FA and FB. Moreover, the electrostatic repulsion between the 4Fe-4S clusters and the positive potential of the backbone atoms is lowest for FX compared to both FA and FB. These results agree with the experimental measurements from the redox titrations of low-temperature EPR signals and of room temperature recombination kinetics.

Cryo-EM structure of a tetrameric photosystem I from Chroococcidiopsis TS-821, a thermophilic, unicellular, non-heterocyst-forming cyanobacterium.

Photosystem I (PSI) is one of two photosystems involved in oxygenic photosynthesis. PSI of cyanobacteria exists in monomeric, trimeric, and tetrameric forms, in contrast to the strictly monomeric form of PSI in plants and algae. The tetrameric organization raises questions about its structural, physiological, and evolutionary significance. Here we report the ∼3.72 Å resolution cryo-electron microscopy structure of tetrameric PSI from the thermophilic, unicellular cyanobacterium Chroococcidiopsis sp. TS-821. The structure resolves 44 subunits and 448 cofactor molecules. We conclude that the tetramer is arranged via two different interfaces resulting from a dimer-of-dimers organization. The localization of chlorophyll molecules permits an excitation energy pathway within and between adjacent monomers. Bioinformatics analysis reveals conserved regions in the PsaL subunit that correlate with the oligomeric state. Tetrameric PSI may function as a key evolutionary step between the trimeric and monomeric forms of PSI organization in photosynthetic organisms.

The Delta Variant Mutations in the Receptor Binding Domain of SARS-CoV-2 Show Enhanced Electrostatic Interactions with the ACE2.

Mutations in the receptor binding domain (RBD) in SARS-CoV-2 are shown to enhance its replication, transmissibility, and binding to host cells. Recently, a new strain is reported in India that includes a mutation (T478K, and L452R) in the RBD, that is possibly increasing the infection rate. Here, using Molecular Mechanics (MM) and Monte Carlo (MC) sampling, we show that the double mutant variant of SARS-CoV-2 induced conformational change in ACE2-E37, which enhanced the electrostatic interactions by the formation of a salt-bridge with SARS-CoV-2-R403. In addition, we observed that the double mutated structure induced a significant change in the salt-bridge electrostatic interaction between RBD-T500 and ACE2-D355. Where that this interaction lost more than 70% of its value compared to its value in WT protein.

The new SARS-CoV-2 strain shows a stronger binding affinity to ACE2 due to N501Y mutant.

SARS-CoV-2 is a global challenge due to its ability to spread much faster than the SARS-CoV, which was attributed to the mutations in the receptor binding domain (RBD). These mutations enhanced the electrostatic interactions. Recently, a new strain is reported in the UK that includes a mutation (N501Y) in the RBD, that is possibly increasing the infection rate. Here, using Molecular Dynamics simulations (MD) and Monte Carlo (MC) sampling, we show that the N501 mutation enhanced the electrostatic interactions due to the formation of a strong hydrogen bond between SARS-CoV-2-T500 and ACE2-D355 near the mutation site. In addition, we observed that the electrostatic interactions between the SARS-CoV-2 and ACE2 in the wild type and the mutant are dominated by salt-bridges formed between SARS-CoV-2-K417 and ACE2-D30, SARS-CoV-2-K458, ACE2-E23, and SARS-CoV-2-R403 and ACE2-E37. These interactions contributed more than 40% of the total binding energies.

ACE2 coding variants in different populations and their potential impact on SARS-CoV-2 binding affinity.

The susceptibility of different populations to SARS-CoV-2 infection is not yet understood. Here, we combined ACE2 coding variants' analysis in different populations and computational chemistry calculations to probe the effects on SARS-CoV-2/ACE2 interaction. ACE2-K26R; which is most frequent in Ashkenazi Jewish population decreased the SARS-CoV-2/ACE2 electrostatic attraction. On the contrary, ACE2-I468V, R219C, K341R, D206G, G211R increased the electrostatic attraction; ordered by binding strength from weakest to strongest. The aforementioned variants are most frequent in East Asian, South Asian, African and African American, European, European and South Asian populations, respectively.

Variations in Proteins Dielectric Constants.

Using a new semi-empirical method for calculating molecular polarizabilities and the Clausius-Mossotti relation, we calculated the static dielectric constants of dry proteins for all structures in the protein data bank (PDB). The mean dielectric constant of more than 150,000 proteins is ϵr=3.23 with a standard deviation of 0.04, which agrees well with previous measurement for dry proteins. The small standard deviation results from the strong correlation between the molecular polarizability and the volume of the proteins. We note that non-amino acid cofactors such as Chlorophyll may alter the dielectric environment significantly. Furthermore, our model shows anisotropies of the dielectric constant within the same molecule according to the constituents amino acids and cofactors. Finally, by changing the amino acid protonation states, we show that a change of pH does not have a significant effect on the dielectric constants of proteins.

Comparing the Binding Interactions in the Receptor Binding Domains of SARS-CoV-2 and SARS-CoV.

SARS-CoV-2, since emerging in Wuhan, China, has been a major concern because of its high infection rate and has left more than six million infected people around the world. Many studies endeavored to reveal the structure of the SARS-CoV-2 compared to the SARS-CoV, in order to find solutions to suppress this high infection rate. Some of these studies showed that the mutations in the SARS-CoV spike (S) protein might be responsible for its higher affinity to the ACE2 human cell receptor. In this work, we used molecular dynamics simulations and Monte Carlo sampling to compare the binding affinities of the S proteins of SARS-CoV and SARS-CoV-2 to the ACE2. Our results show that the protein surface of the ACE2 at the receptor binding domain (RBD) exhibits negative electrostatic potential, while a positive potential is observed for the S proteins of SARS-CoV/SARS-CoV-2. In addition, the binding energies at the interface are slightly higher for SARS-CoV-2 because of enhanced electrostatic interactions. The major contributions to the electrostatic binding energies result from the salt bridges forming between R426 and ACE-2-E329 in the case of SARS-CoV and K417 and ACE2-D30 in the SARS-CoV-2. In addition, our results indicate that the enhancement in the binding energy is not due to a single mutant but rather because of the sophisticated structural changes induced by all these mutations together. This finding suggests that it is implausible for the SARS-CoV-2 to be a lab-engineered virus.

Controlled beams of shock-frozen, isolated, biological and artificial nanoparticles.

X-ray free-electron lasers promise diffractive imaging of single molecules and nanoparticles with atomic spatial resolution. This relies on the averaging of millions of diffraction patterns of identical particles, which should ideally be isolated in the gas phase and preserved in their native structure. Here, we demonstrated that polystyrene nanospheres and Cydia pomonella granulovirus can be transferred into the gas phase, isolated, and very quickly shock-frozen, i.e., cooled to 4 K within microseconds in a helium-buffer-gas cell, much faster than state-of-the-art approaches. Nanoparticle beams emerging from the cell were characterized using particle-localization microscopy with light-sheet illumination, which allowed for the full reconstruction of the particle beams, focused to < 100 µ m , as well as for the determination of particle flux and number density. The experimental results were quantitatively reproduced and rationalized through particle-trajectory simulations. We propose an optimized setup with cooling rates for particles of few-nanometers on nanosecond timescales. The produced beams of shock-frozen isolated nanoparticles provide a breakthrough in sample delivery, e.g., for diffractive imaging and microscopy or low-temperature nanoscience.

Probing the Water Stability Limits and Degradation Pathways of Metal-Organic Frameworks.

A comprehensive model to describe the water stability of prototypical metal-organic frameworks (MOFs) is derived by combining different types of theoretical and experimental approaches. The results provide an insight into the early stages of water-triggered destabilization of MOFs and allow detailed pathways to be proposed for the degradation of different MOFs under aqueous conditions. The essential elements of the approach are computing the pKa values of coordinated water molecules and geometry relaxations. Variable-temperature and pH infrared spectroscopy techniques are used to corroborate the main findings. The model developed herein helps to explain stability limits observed for several prototypical MOFs, including MOF-5, HKUST-1, UiO-66, and MIL-101-Cr, in aqueous solutions, and thus, provides an insight into the possible degradation pathways in acidic and basic environments. The formation of a metal hydroxide through the autoprotolysis of metal-coordinated water molecules and the strength of carboxylate-metal interactions are suggested to be two key players that govern stability in basic and acidic media, respectively. The methodology presented herein can effectively guide future efforts, which are especially significant for in silico screening, for developing novel MOFs with enhanced aqueous stability.

Tuning the Chemical Environment within the UiO-66-NH2 Nanocages for Charge-Dependent Contaminant Uptake and Selectivity.

The remarkable water stability of Zr-carboxylate-based metal-organic frameworks (MOFs) stimulated considerable interest toward their utilization in aqueous phase applications. The origin of such stability is probed here through pH titration and pKa modeling. A unique feature of the Zr6(µ3-OH)4(µ3-O)4(RCO2)12 cluster is the Zr-bridging oxo/hydroxyl groups, demonstrating several pKa values that appear to provide for the water stability at a wide range of pH. Accordingly, the tunability of the cage/surface charge of the MOF can feasibly be controlled through careful adjustment of solution pH. Such high stability, and facile control over cage/surface charge, can additionally be augmented through introducing chemical functionalities lining the cages of the MOF, specifically amine groups in the UiO-66-NH2 presented herein. The variable protonation states of the Zr cluster and the pendant amino groups, their H-bond donor/acceptor characteristics, and their electrostatic interactions with guest molecules were effectively utilized in controlled experiments to demonstrate high uptake of model guest molecules (137 mg/g for Cr(VI), 1275 mg/g for methylene blue, and 909 mg/g for methyl orange). Additionally, a practical form of the silica-supported MOF, UiO-66-NH2@SiO2, constructed in under 2 h reaction time, is described, generating a true platform microporous sorbent for practical use in demanding applications.

Thermodynamics of the S2-to-S3 state transition of the oxygen-evolving complex of photosystem II.

The room temperature pump-probe X-ray free electron laser (XFEL) measurements used for serial femtosecond crystallography provide remarkable information about the structures of the catalytic (S-state) intermediates of the oxygen-evolution reaction of photosystem II. However, mixed populations of these intermediates and moderate resolution limit the interpretation of the data from current experiments. The S3 XFEL structures show extra density near the OEC that may correspond to a water/hydroxide molecule. However, in the latest structure, this additional oxygen is 2.08 Šfrom the Oε2 of D1-E189, which is closer than the sum of the van der Waals radii of the two oxygens. Here, we use Boltzmann statistics and Monte Carlo sampling to provide a model for the S2-to-S3 state transition, allowing structural changes and the insertion of an additional water/hydroxide. Based on our model, water/hydroxide addition to the oxygen-evolving complex (OEC) is not thermodynamically favorable in the S2g = 2 state, but it is in the S2g = 4.1 redox isomer. Thus, formation of the S3 state starts by a transition from the S2g = 2 to the S2g = 4.1 structure. Then, electrostatic interactions support protonation of D1-H190 and deprotonation of the Ca2+-ligated water (W3) with proton loss to the lumen. The W3 hydroxide moves toward Mn4, completing the coordination shell of Mn4 and favoring its oxidation to Mn(iv) in the S3 state. In addition, binding an additional hydroxide to Mn1 leads to a conformational change of D1-E189 in the S2g = 4.1 and S3 structures. In the S3 state a fraction of D1-E189 release from Mn1 and bind a proton.

Robust and Accurate Computational Estimation of the Polarizability Tensors of Macromolecules.

Alignment of molecules through electric fields minimizes the averaging over orientations, e.g., in single-particle-imaging experiments. The response of molecules to external ac electric fields is governed by their polarizability tensor, which is usually calculated using quantum chemistry methods. These methods are not feasible for large molecules. Here, we calculate the polarizability tensor of proteins using a regression model that correlates the polarizabilities of the 20 amino acids with perfect conductors of the same shape. The dielectric constant of the molecules could be estimated from the slope of the regression line based on the Clausius-Mossotti equation. We benchmark our predictions against the quantum chemistry results for the Trp cagemini protein and the measured dielectric constants of larger proteins. Our method has applications in computing laser alignment of macromolecules, for instance, benefiting single-particle imaging, as well as for estimation of the optical and electrostatic characteristics of proteins and other macromolecules.

Relative stability of the S2 isomers of the oxygen evolving complex of photosystem II.

The oxidation of water to O2 is catalyzed by the Oxygen Evolving Complex (OEC), a Mn4CaO5 complex in Photosystem II (PSII). The OEC is sequentially oxidized from state S0 to S4. The S2 state, (MnIII)(MnIV)3, coexists in two redox isomers: S2,g=2, where Mn4 is MnIV and S2,g=4.1, where Mn1 is MnIV. Mn4 has two terminal water ligands, whose proton affinity is affected by the Mn oxidation state. The relative energy of the two S2 redox isomers and the protonation state of the terminal water ligands are analyzed using classical multi-conformer continuum electrostatics (MCCE). The Monte Carlo simulations are done on QM/MM optimized S1 and S2 structures docked back into the complete PSII, keeping the protonation state of the protein at equilibrium with the OEC redox and protonation states. Wild-type PSII, chloride-depleted PSII, PSII in the presence of oxidized YZ/protonated D1-H190, and the PSII mutants D2-K317A, D1-D61A, and D1-S169A are studied at pH 6. The wild-type PSII at pH 8 is also described. In qualitative agreement with experiment, in wild-type PSII, the S2,g=2 redox isomer is the lower energy state; while chloride depletion or pH 8 stabilizes the S2,g=4.1 state and the mutants D2-K317A, D1-D61A, and D1-S169A favor the S2,g=2 state. The protonation states of D1-E329, D1-E65, D1-H337, D1-D61, and the terminal waters on Mn4 (W1 and W2) are affected by the OEC oxidation state. The terminal W2 on Mn4 is a mixture of water and hydroxyl in the S2,g=2 state, indicating the two water protonation states have similar energy, while it remains neutral in the S1 and S2,g=4.1 states. In wild-type PSII, advancement to S2 leads to negligible proton loss and so there is an accumulation of positive charge. In the analyzed mutations and Cl- depleted PSII, additional deprotonation is found upon formation of S2 state.