Cofactor Dynamics Couples the Protein Surface to the Heme in Cytochrome c, Facilitating Electron Transfer.

Electron transport through biomolecules and in biological transport networks is of great importance to bioenergetics and biocatalysis. More generally, it is of crucial importance to understand how the pathways that connect buried metallocofactors to other cofactors, and to protein surfaces, affect the biological chemistry of metalloproteins. In terms of electron transfer (ET), the strongest coupling pathways usually comprise covalent and hydrogen bonded networks, with a limited number of through-space contacts. Herein, we set out to determine the relative roles of hydrogen bonds involved in ET via an established heme-to-surface tunneling pathway in cytochrome (cyt) c (i.e., heme-W59-D60-E61-N62). A series of cyt c variants were produced where a ruthenium tris(diimine) photooxidant was placed at position 62 via covalent modification of the N62C residue. Surprisingly, variants where the H-bonding residues W59 and D60 were replaced (i.e., W59F and D60A) showed no change in ET rate from the ferrous heme to Ru(III). In contrast, changing the composition of an alternative tunneling pathway (i.e., heme-M64-N63-C62) with the M64L substitution shows a factor of 2 decrease in the rate of heme-to-Ru ET. This pathway involves a through-space tunneling step between the heme and M64 residue, and such steps are usually disfavored. To rationalize why the heme-M64-N63-C62 is preferred, molecular dynamics (MD) simulations and Pathways analysis were employed. These simulations show that the change in heme-Ru ET rates is attributed to different conformations with compressed donor-acceptor distances, by ∼2 Å in pathway distance, in the M64-containing protein as compared to the M64L protein. The change in distance is correlated with changes in the electronic coupling that are in accord with the experimentally observed heme-Ru ET rates. Remarkably, the M64L variation at the core of the protein translates to changes in cofactor dynamics at the protein surface. The surface changes identified by MD simulations include dynamic anion-π and dipole-dipole interactions. These interactions influence the strength of tunneling pathways and ET rates by facilitating decreases in through-space tunneling distances in key coupling pathways.

Machine Learning Approaches for Metalloproteins.

Metalloproteins are a family of proteins characterized by metal ion binding, whereby the presence of these ions confers key catalytic and ligand-binding properties. Due to their ubiquity among biological systems, researchers have made immense efforts to predict the structural and functional roles of metalloproteins. Ultimately, having a comprehensive understanding of metalloproteins will lead to tangible applications, such as designing potent inhibitors in drug discovery. Recently, there has been an acceleration in the number of studies applying machine learning to predict metalloprotein properties, primarily driven by the advent of more sophisticated machine learning algorithms. This review covers how machine learning tools have consolidated and expanded our comprehension of various aspects of metalloproteins (structure, function, stability, ligand-binding interactions, and inhibitors). Future avenues of exploration are also discussed.

Modulation of Phospholipid Bilayer Properties by Simvastatin.

Simvastatin (Zocor) is one of the most prescribed drugs for reducing high cholesterol. Although simvastatin is ingested in its inactive lactone form, it is converted to its active dihydroxyheptanoate form by carboxylesterases in the liver. The dihydroxyheptanoate form can also be converted back to its original lactone form. Unfortunately, some of the side effects associated with the intake of simvastatin and other lipophilic statins at higher doses include statin-associated myopathy (SAM) and, in more severe cases, kidney failure. While the cause of SAM is unknown, it is hypothesized that these side effects are dependent on the localization of statins in lipid bilayers and their impact on bilayer properties. In this work, we carry out all-atom molecular dynamics simulations on both the lactone and dihydroxyheptanoate forms of simvastatin (termed "SN" and "SA", respectively) with a pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer and a POPC/cholesterol (30 mol %) binary mixture as membrane models. Additional simulations were carried out with multiple simvastatin molecules to mimic in vitro conditions that produced pleiotropic effects. Both SN and SA spontaneously diffused into the lipid bilayer, and a longer simulation time of 4 µs was needed for the complete incorporation of multiple SAs into the bilayer. By constructing potential mean force and electron density profiles, we find that SN localizes deeper within the hydrophobic interior of the bilayer and that SA has a greater tendency to form hydrogen-bonding interactions with neighboring water molecules and lipid headgroups. For the pure POPC bilayer, both SN and SA increase membrane order, while membrane fluidity increases for the POPC/cholesterol bilayer.

Oxalate decarboxylase uses electron hole hopping for catalysis.

The hexameric low-pH stress response enzyme oxalate decarboxylase catalyzes the decarboxylation of the oxalate mono-anion in the soil bacterium Bacillus subtilis. A single protein subunit contains two Mn-binding cupin domains, and catalysis depends on Mn(III) at the N-terminal site. The present study suggests a mechanistic function for the C-terminal Mn as an electron hole donor for the N-terminal Mn. The resulting spatial separation of the radical intermediates directs the chemistry toward decarboxylation of the substrate. A π-stacked tryptophan pair (W96/W274) links two neighboring protein subunits together, thus reducing the Mn-to-Mn distance from 25.9 Å (intrasubunit) to 21.5 Å (intersubunit). Here, we used theoretical analysis of electron hole-hopping paths through redox-active sites in the enzyme combined with site-directed mutagenesis and X-ray crystallography to demonstrate that this tryptophan pair supports effective electron hole hopping between the C-terminal Mn of one subunit and the N-terminal Mn of the other subunit through two short hops of ∼8.5 Å. Replacement of W96, W274, or both with phenylalanine led to a large reduction in catalytic efficiency, whereas replacement with tyrosine led to recovery of most of this activity. W96F and W96Y mutants share the wildtype tertiary structure. Two additional hole-hopping networks were identified leading from the Mn ions to the protein surface, potentially protecting the enzyme from high Mn oxidation states during turnover. Our findings strongly suggest that multistep hole-hopping transport between the two Mn ions is required for enzymatic function, adding to the growing examples of proteins that employ aromatic residues as hopping stations.

Temperature Dependence of Charge and Spin Transfer in Azurin.

The steady-state charge and spin transfer yields were measured for three different Ru-modified azurin derivatives in protein films on silver electrodes. While the charge-transfer yields exhibit weak temperature dependences, consistent with operation of a near activation-less mechanism, the spin selectivity of the electron transfer improves as temperature increases. This enhancement of spin selectivity with temperature is explained by a vibrationally induced spin exchange interaction between the Cu(II) and its chiral ligands. These results indicate that distinct mechanisms control charge and spin transfer within proteins. As with electron charge transfer, proteins deliver polarized electron spins with a yield that depends on the protein's structure. This finding suggests a new role for protein structure in biochemical redox processes.

Evaluation of all-atom force fields in viral capsid simulations and properties.

As the past century has been characterized by waves of viral pandemics, there is an ever-growing role for molecular simulation-based research. In this study, we utilize all-atom molecular dynamics to simulate an enterovirus-D68 capsid and examine the dependency of viral capsid dynamics and properties on AMBER and CHARMM force fields. Out of the six force fields studied, we note that CHARMM36m and CHARMM36 generate secondary structures that are most consistent with protein structural data and sample the largest conformational space. The ion distribution and radius of gyration of the capsid are similar across all force fields investigated.

Correlation between Charge Transport and Base Excision Repair in the MutY-DNA Glycosylase.

Experimental evidence suggests that DNA-mediated redox signaling between high-potential [Fe4S4] proteins is relevant to DNA replication and repair processes, and protein-mediated charge transfer (CT) between [Fe4S4] clusters and nucleic acids is a fundamental process of the signaling and repair mechanisms. We analyzed the dominant CT pathways in the base excision repair glycosylase MutY using molecular dynamics simulations and hole hopping pathway analysis. We find that the adenine nucleobase of the mismatched A·oxoG DNA base pair facilitates [Fe4S4]-DNA CT prior to adenine excision by MutY. We also find that the R153L mutation in MutY (linked to colorectal adenomatous polyposis) influences the dominant [Fe4S4]-DNA CT pathways and appreciably decreases their effective CT rates.

Mutation effects on charge transport through the p58c iron-sulfur protein.

Growing experimental evidence indicates that iron-sulfur proteins play key roles in DNA repair and replication. In particular, charge transport between [Fe4S4] clusters, mediated by proteins and DNA, may convey signals to coordinate enzyme action. Human primase is a well studied [Fe4S4] protein, and its p58c domain (which contains an [Fe4S4] cluster) plays a role in the initiation of DNA replication. The Y345C mutation in p58c is linked to gastric tumors and may influence the protein-mediated charge transport. The complexity of protein-DNA systems, and the intricate electronic structure of [Fe4S4] clusters, have impeded progress into understanding functional charge transport in these systems. In this study, we built force fields to describe the high potential [Fe4S4] cluster in both oxidation states. The parameterization is compatible with AMBER force fields and enabled well-balanced molecular dynamics simulations of the p58c-RNA/DNA complex relevant to the initiation of DNA replication. Using the molecular mechanics Poisson-Boltzmann and surface area solvation method on the molecular dynamics trajectories, we find that the p58c mutation induces a modest change in the p58c-duplex binding free energy in agreement with recent experiments. Through kinetic modeling and analysis, we identify key features of the main charge transport pathways in p58c. In particular, we find that the Y345C mutation partially changes the composition and frequency of the most efficient (and potentially relevant to the biological function) charge transport pathways between the [Fe4S4] cluster and the duplex. Moreover, our approach sets the stage for a deeper understanding of functional charge transfer in [Fe4S4] protein-DNA complexes.

2'-Deoxy-2'-fluoro-arabinonucleic acid: a valid alternative to DNA for biotechnological applications using charge transport.

The non-biological 2'-deoxy-2'-fluoro-arabinonucleic acid (2'F-ANA) may be used as a valid alternative to DNA in biomedical and electronic applications because of its higher resistance to hydrolysis and nuclease degradation. However, the advantage of using 2'F-ANA in such applications also depends on its charge-transfer properties compared to DNA. In this study, we compare the charge conduction properties of model 2'F-ANA and DNA double-strands, using structural snapshots from MD simulations to calculate the electronic couplings and reorganization energies associated with the hole transfer steps between adjacent nucleobase pairs. Inserting these charge-transfer parameters into a kinetic model for charge conduction, we find similar conductive properties for DNA and 2'F-ANA. Moreover, we find that 2'F-ANA's enhanced chemical stability does not correspond to a reduction in the nucleobase π-stack structural flexibility relevant to both electronic couplings and reorganization free energies. Our results promote the use of 2'F-ANA in applications that can be based on charge transport, such as biosensing and chip technology, where its chemical stability and conductivity can advantageously combine.

Correction: Charge Transfer between [4Fe4S] Proteins and DNA Is Unidirectional: Implications for Biomolecular Signaling.

[This corrects the article PMC6350243.].

Mapping hole hopping escape routes in proteins.

A recently proposed oxidative damage protection mechanism in proteins relies on hole hopping escape routes formed by redox-active amino acids. We present a computational tool to identify the dominant charge hopping pathways through these residues based on the mean residence times of the transferring charge along these hopping pathways. The residence times are estimated by combining a kinetic model with well-known rate expressions for the charge-transfer steps in the pathways. We identify the most rapid hole hopping escape routes in cytochrome P450 monooxygenase, cytochrome c peroxidase, and benzylsuccinate synthase (BSS). This theoretical analysis supports the existence of hole hopping chains as a mechanism capable of providing hole escape from protein catalytic sites on biologically relevant timescales. Furthermore, we find that pathways involving the [4Fe4S] cluster as the terminal hole acceptor in BSS are accessible on the millisecond timescale, suggesting a potential protective role of redox-active cofactors for preventing protein oxidative damage.

Charge Transfer between [4Fe4S] Proteins and DNA Is Unidirectional: Implications for Biomolecular Signaling.

Recent experiments suggest that DNA-mediated charge transport might enable signaling between the [4Fe4S] clusters in the C-terminal domains of human DNA primase and polymerase α, as well as the signaling between other replication and repair high-potential [4Fe4S] proteins. Our theoretical study demonstrates that the redox signaling cannot be accomplished exclusively by DNA-mediated charge transport because part of the charge transfer chain has an unfavorable free energy profile. We show that hole or excess electron transfer between a [4Fe4S] cluster and a nucleic acid duplex through a protein medium can occur within microseconds in one direction, while it is kinetically hindered in the opposite direction. We present a set of signaling mechanisms that may occur with the assistance of oxidants or reductants, using the allowed charge transfer processes. These mechanisms would enable the coordinated action of [4Fe4S] proteins on DNA, engaging the [4Fe4S] oxidation state dependence of the protein-DNA binding affinity.

Cell-Penetrating Protein/Corrole Nanoparticles.

Recent work has highlighted the potential of metallocorroles as versatile platforms for the development of drugs and imaging agents, since the bioavailability, physicochemical properties and therapeutic activity can be dramatically altered by metal ion substitution and/or functional group replacement. Significant advances in cancer treatment and imaging have been reported based on work with a water-soluble bis-sulfonated gallium corrole in both cellular and rodent-based models. We now show that cytotoxicities increase in the order Ga < Fe < Al < Mn < Sb < Au for bis-sulfonated corroles; and, importantly, that they correlate with metallocorrole affinities for very low density lipoprotein (VLDL), the main carrier of lipophilic drugs. As chemotherapeutic potential is predicted to be enhanced by increased lipophilicity, we have developed a novel method for the preparation of cell-penetrating lipophilic metallocorrole/serum-protein nanoparticles (NPs). Cryo-TEM revealed an average core metallocorrole particle size of 32 nm, with protein tendrils extending from the core (conjugate size is ~100 nm). Optical imaging of DU-145 prostate cancer cells treated with corrole NPs (≤100 nM) revealed fast cellular uptake, very slow release, and distribution into the endoplasmic reticulum (ER) and lysosomes. The physical properties of corrole NPs prepared in combination with transferrin and albumin were alike, but the former were internalized to a greater extent by the transferrin-receptor-rich DU-145 cells. Our method of preparation of corrole/protein NPs may be generalizable to many bioactive hydrophobic molecules to enhance their bioavailability and target affinity.

A single AT-GC exchange can modulate charge transfer-induced p53-DNA dissociation.

Using molecular dynamics simulations and electronic structure theory, we shed light on the charge dynamics that causes the differential interaction of tumor suppressor protein p53 with the p21 and Gadd45 genes in response to oxidative stress. We show that the sequence dependence of this selectivity results from competing charge transfer to the protein and through the DNA, with implications on the use of genome editing tools to influence the p53 regulatory function.

Electron transfer characteristics of 2'-deoxy-2'-fluoro-arabinonucleic acid, a nucleic acid with enhanced chemical stability.

The non-biological nucleic acid 2'-deoxy-2'-fluoro-arabinonucleic acid (2'F-ANA) may be of use because of its higher chemical stability than DNA in terms of resistance to hydrolysis and nuclease degradation. In order to investigate the charge transfer characteristics of 2'F-ANA, of relevance to applications in nucleic acid-based biosensors and chip technologies, we compare the electronic couplings for hole transfer between stacked nucleobase pairs in DNA and 2'F-ANA by carrying out density functional theory (DFT) calculations on geometries taken from molecular dynamics simulations. We find similar averages and distribution widths of the base-pair couplings in the two systems. On the basis of this result, 2'F-ANA is expected to have charge transfer properties similar to those of DNA, while offering the advantage of enhanced chemical stability. As such, 2'F-ANA may serve as a possible alternative to DNA for use in a broad range of nanobiotechnological applications. Furthermore, we show that the (experimentally observed) enhanced chemical stability resulting from the backbone modifications does not cause reduced fluctuations of the base-pair electronic couplings around the values found for "ideal" B-DNA (with standard step parameter values). Our study also supports the use of a DFT implementation, with the M11 functional, of the wave function overlap method to compute effective electronic couplings in nucleic acid systems.

Fighting Cancer with Corroles.

Corroles are exceptionally promising platforms for the development of agents for simultaneous cancer-targeting imaging and therapy. Depending on the element chelated by the corrole, these theranostic agents may be tuned primarily for diagnostic or therapeutic function. Versatile synthetic methodologies allow for the preparation of amphipolar derivatives, which form stable noncovalent conjugates with targeting biomolecules. These conjugates can be engineered for imaging and targeting as well as therapeutic function within one theranostic assembly. In this review, we begin with a brief outline of corrole chemistry that has been uniquely useful in designing corrole-based anticancer agents. Then we turn attention to the early literature regarding corrole anticancer activity, which commenced one year after the first scalable synthesis was reported (1999-2000). In 2001, a major advance was made with the introduction of negatively charged corroles, as these molecules, being amphipolar, form stable conjugates with many proteins. More recently, both cellular uptake and intracellular trafficking of metallocorroles have been documented in experimental investigations employing advanced optical spectroscopic as well as magnetic resonance imaging techniques. Key results from work on both cellular and animal models are reviewed, with emphasis on those that have shed new light on the mechanisms associated with anticancer activity. In closing, we predict a very bright future for corrole anticancer research, as it is experiencing exponential growth, taking full advantage of recently developed imaging and therapeutic modalities.

Lanthanides: Applications in Cancer Diagnosis and Therapy.

Lanthanide complexes are of increasing importance in cancer diagnosis and therapy, owing to the versatile chemical and magnetic properties of the lanthanide-ion 4f electronic configuration. Following the first implementation of gadolinium(III)-based contrast agents in magnetic resonance imaging in the 1980s, lanthanide-based small molecules and nanomaterials have been investigated as cytotoxic agents and inhibitors, in photodynamic therapy, radiation therapy, drug/gene delivery, biosensing, and bioimaging. As the potential utility of lanthanides in these areas continues to increase, this timely review of current applications will be useful to medicinal chemists and other investigators interested in the latest developments and trends in this emerging field.

Computational predictions of corroles as a class of Hsp90 inhibitors.

Corroles have been shown experimentally to cause cell cycle arrest, and there is some evidence that this might be attributed to an inhibitory effect of corroles on Heat shock protein 90 (Hsp90), which is known to play a vital role in cancer cell proliferation. In this study, we used molecular dynamics to examine the interaction of gallium corroles with Hsp90, and found that they can bind preferentially to the ATP-binding N-terminal site. We also found that structural variations of the corrole ring can influence the binding energies and affinities of the corrole to Hsp90. We predict that both the bis-carboxylated corrole (4-Ga) and a proposed 3,17-bis-sulfonated corrole (7-Ga) are promising alternatives to Ga(III) 5,10,15-tris(pentafluorophenyl)-2,17-bis(sulfonic acid)-corrole (1-Ga) as anti-cancer agents.

A cytotoxic and cytostatic gold(III) corrole.

We have synthesized and characterized a water-soluble gold(III) corrole (1-Au) that is highly toxic to cisplatin-resistant cancer cells. Relative to its 1-Ga analogue, axial ligands bind only weakly to 1-Au, which likely accounts for its lower affinity for human serum albumin (HSA). We suggest that the cytotoxicity of 1-Au may be related to this lower HSA affinity.