Deriving Immune-Modulating Peptides from Viral Serine Protease Inhibitors (Serpins).

Viruses have devised highly effective approaches that modulate the host immune response, blocking immune responses that are designed to eradicate viral infections. Over millions of years of evolution, virus-derived immune-modulating proteins have become extraordinarily potent, in some cases working at picomolar concentrations when expressed into surrounding tissues and effectively blocking host defenses against viral invasion and replication. The marked efficiency of these immune-modulating proteins is postulated to be due to viral engineering of host immune modulators as well as design and development of new strategies (i.e., some derived from host proteins and some entirely unique). Two key characteristics of viral immune modulators confer both adaptive advantages and desirable functions for therapeutic translation. First, many virus-derived immune modulators have evolved structures that are not readily recognized or regulated by mammalian immune pathways, ensuring little to no neutralizing antibody responses or proteasome-mediated degradation. Second, these immune modulators tend to target early steps in central immune responses, producing a powerful downstream inhibitory "domino effect" which may alter cell activation and gene expression.We have proposed that peptide metabolites of these immune-modulating proteins can enhance and extend protein function. Active immunomodulating peptides have been derived from both mammalian and viral proteins. We previously demonstrated that peptides derived from computationally predicted cleavage sites in the reactive center loop (RCL) of a viral serine proteinase inhibitor (serpin ) from myxoma virus, Serp-1 , can modify immune response activation. We have also demonstrated modulation of host gut microbiota produced by Serp-1 and RCL-derived peptide , S7, in a vascular inflammation model. Of interest, generation of derived peptides that maintain therapeutic function from a serpin can act by a different mechanism. Whereas Serp-1 has canonical serpin-like function to inhibit serine proteases, S7 instead targets mammalian serpins. Here we describe the derivation of active Serp- RCL peptides and their testing in inflammatory vasculitis models.

Immune protection is dependent on the gut microbiome in a lethal mouse gammaherpesviral infection.

Immunopathogenesis in systemic viral infections can induce a septic state with leaky capillary syndrome, disseminated coagulopathy, and high mortality with limited treatment options. Murine gammaherpesvirus-68 (MHV-68) intraperitoneal infection is a gammaherpesvirus model for producing severe vasculitis, colitis and lethal hemorrhagic pneumonia in interferon gamma receptor-deficient (IFNγR-/-) mice. In prior work, treatment with myxomavirus-derived Serp-1 or a derivative peptide S-7 (G305TTASSDTAITLIPR319) induced immune protection, reduced disease severity and improved survival after MHV-68 infection. Here, we investigate the gut bacterial microbiome in MHV-68 infection. Antibiotic suppression markedly accelerated MHV-68 pathology causing pulmonary consolidation and hemorrhage, increased mortality and specific modification of gut microbiota. Serp-1 and S-7 reduced pulmonary pathology and detectable MHV-68 with increased CD3 and CD8 cells. Treatment efficacy was lost after antibiotic treatments with associated specific changes in the gut bacterial microbiota. In summary, transkingdom host-virus-microbiome interactions in gammaherpesvirus infection influences gammaherpesviral infection severity and reduces immune modulating therapeutic efficacy.

Molecular Modeling-Guided Design of Phospholipid-Based Prodrugs.

The lipidic prodrug approach is an emerging field for improving a number of biopharmaceutical and drug delivery aspects. Owing to their structure and nature, phospholipid (PL)-based prodrugs may join endogenous lipid processing pathways, and hence significantly improve the pharmacokinetics and/or bioavailability of the drug. Additional advantages of this approach include drug targeting by enzyme-triggered drug release, blood-brain barrier permeability, lymphatic targeting, overcoming drug resistance, or enabling appropriate formulation. The PL-prodrug design includes various structural modalities-different conjugation strategies and/or the use of linkers between the PL and the drug moiety, which considerably influence the prodrug characteristics and the consequent effects. In this article, we describe how molecular modeling can guide the structural design of PL-based prodrugs. Computational simulations can predict the extent of phospholipase A2 (PLA2)-mediated activation, and facilitate prodrug development. Several computational methods have been used to facilitate the design of the pro-drugs, which will be reviewed here, including molecular docking, the free energy perturbation method, molecular dynamics simulations, and free density functional theory. Altogether, the studies described in this article indicate that computational simulation-guided PL-based prodrug molecular design correlates well with the experimental results, allowing for more mechanistic and less empirical development. In the future, the use of molecular modeling techniques to predict the activity of PL-prodrugs should be used earlier in the development process.

Phospholipid-Based Prodrugs for Colon-Targeted Drug Delivery: Experimental Study and In-Silico Simulations.

In ulcerative colitis (UC), the inflammation is localized in the colon, and one of the successful strategies for colon-targeting drug delivery is the prodrug approach. In this work, we present a novel phospholipid (PL)-based prodrug approach, as a tool for colonic drug targeting in UC. We aim to use the phospholipase A2 (PLA2), an enzyme that is overexpressed in the inflamed colonic tissues of UC patients, as the PL-prodrug activating enzyme, to accomplish the liberation of the parent drug from the prodrug complex at the specific diseased tissue(s). Different linker lengths between the PL and the drug moiety can dictate the rate of activation by PLA2, and subsequently determine the amount of free drugs at the site of action. The feasibility of this approach was studied with newly synthesized PL-Fmoc (fluorenylmethyloxycarbonyl) conjugates, using Fmoc as a model compound for testing our hypothesis. In vitro incubation with bee venom PLA2 demonstrated that a 7-carbon linker between the PL and Fmoc has higher activation rate than a 5-carbon linker. 4-fold higher colonic expression of PLA2 was demonstrated in colonic mucosa of colitis-induced rats when compared to healthy animals, validating our hypothesis of a colitis-targeting prodrug approach. Next, a novel molecular dynamics (MD) simulation was developed for PL-based prodrugs containing clinically relevant drugs. PL-methotrexate conjugate with 6-carbon linker showed the highest extent of PLA2-mediated activation, whereas shorter linkers were activated to a lower extent. In conclusion, this work demonstrates that for carefully designed PL-drug conjugates, PLA2 overexpression in inflamed colonic tissues can be used as prodrug-activating enzyme and drug targeting strategy, including insights into the activation mechanisms in a PLA2 binding site.

Lipidic prodrug approach for improved oral drug delivery and therapy.

In the past, a prodrug design was used as a last option to improve bioavailability through controlling transport, distribution, metabolism, or other mechanisms. Prodrugs are currently used even in early stages of drug development, and a significant percentage of all drugs in the market are prodrugs. The focus of this article is lipidic prodrugs, a strategy whereby a lipid carrier is covalently bound to the drug moiety. The increased lipophilicity of the lipid-drug conjugate can improve the pharmacokinetic profile and provide meaningful advantages: increased absorption across biological barriers, prolonged circulation half-life, selective distribution profile (eg brain penetration), reduced hepatic first-pass metabolism, and overall enhanced bioavailability of the parent drug. Moreover, lipidic prodrugs may join the endogenous lipid trafficking pathways, thereby facilitate drug targeting, either by selective absorption pathway (eg lymphatic transport) or drug release at specific target site(s). The different lipid-drug conjugates (triglyceride-, fatty acids, phospholipid-, and steroid-based prodrugs), the physiological barriers that challenge the absorption of these conjugates, followed by their current utilization and potential clinical benefits are described and analyzed, and future opportunities this approach could provide are discussed. Altogether, lipidic prodrugs represent an exciting approach for improving different aspects of oral drug delivery/therapy and may provide solutions for various unmet needs; the use of this strategy is expected to grow.

Prospects and Challenges of Phospholipid-Based Prodrugs.

Nowadays, the prodrug approach is used already at the early stages of drug development. Lipidic prodrug approach is a growing field for improving a number of drug properties/delivery/therapy aspects, and can offer solutions for various unmet needs. This approach includes drug moiety bound to the lipid carrier, which can be triglyceride, fatty acids, steroid, or phospholipid (PL). The focus of this article is PL-based prodrugs, which includes a PL carrier covalently bound to the active drug moiety. An overview of relevant physiological lipid processing pathways and absorption barriers is provided, followed by drug delivery/therapeutic application of PL-drug conjugates, as well as computational modeling techniques, and a modern bioinformatics tool that can aid in the optimization of PL conjugates. PL-based prodrugs have increased lipophilicity comparing to the parent drug, and can therefore significantly improve the pharmacokinetic profile and overall bioavailability of the parent drug, join the endogenous lipid processing pathways and therefore accomplish drug targeting, e.g., by lymphatic transport, drug release at specific target site(s), or passing the blood-brain barrier. Moreover, an exciting gateway for treating inflammatory diseases and cancer is presented, by utilizing the PL sn-2 position in the prodrug design, aiming for PLA2-mediated activation. Overall, a PL-based prodrug approach shows great potential in improving different drug delivery/therapy aspects, and is expected to grow.

Activation of the ß-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance and arterial blood pressure. However, EPO stimulates the production of the potent vasodilator, nitric oxide (NO), in culture endothelial cells. The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to activate two receptor complexes, the homodimeric EPO (EPOR2 ) and the heterodimeric EPOR/ß-common receptor (ßCR). The purpose of this study was to investigate the contribution of each receptor to the vasoactive properties of EPO. First-order, mesenteric arteries were isolated from 16-week-old male C57BL/6 mice, and arterial function was studied in pressure arteriographs. To determine the contribution of each receptor complex, EPO-stimulating peptide (ESP), which binds and activates the heterodimeric EPOR/ßCR complex, and EPO, which activates both receptors, were added to the arteriograph chamber 20 min prior to evaluation of endothelium-dependent (acetylcholine, bradykinin, A23187) and endothelium-independent (sodium nitroprusside) vasodilator responses. Only ACh-induced vasodilation was impaired in arteries pretreated with EPO or ESP. EPO and ESP pretreatment abolished ACh-induced vasodilation by 100% and 60%, respectively. EPO and ESP did not affect endothelium-independent vasodilation by SNP. Additionally, a novel ßCR inhibitory peptide (ßIP), which was computationally developed, prevented the impairment of acetylcholine-induced vasodilation by EPO and ESP, further implicating the EPOR/ßCR complex. Last, pretreatment with either EPO or ESP did not affect vasoconstriction by phenylephrine and KCl. Taken together, these findings suggest that acute activation of the heterodimeric EPOR/ßCR in endothelial cells leads to a selective impairment of ACh-mediated vasodilator response in mouse mesenteric resistance arteries.

Computational design and experimental characterization of a novel ß-common receptor inhibitory peptide.

In short-term animal models of ischemia, erythropoietin (EPO) signaling through the heterodimeric EPO receptor (EPOR)/ß-common receptor (ßCR) is believed to elicit tissue protective effects. However, large, randomized, controlled trials demonstrate that targeting a higher hemoglobin level by administering higher doses of EPO, which are more likely to activate the heterodimeric EPOR/ßCR, is associated with an increase in adverse cardiovascular events. Thus, inhibition of long-term activation of the ßCR may have therapeutic implications. This study aimed to design and evaluate the efficacy of novel computationally designed ßCR inhibitory peptides (ßIP). These novel ßIPs were designed based on a truncated portion of Helix-A from EPO, specifically residues 11-26 (VLERYLLEAKEAEKIT). Seven novel peptides (P1 to P7) were designed. Peptide 7 (P7), VLERYLHEAKHAEKIT, demonstrated the most robust inhibitory activity. We also report here the ability of P7 to inhibit ßCR-induced nitric oxide (NO) production and angiogenesis in human umbilical vein endothelial cells (HUVECs). Specifically, we found that P7 ßIP completely abolished EPO-induced NO production. The inhibitory effect could be overcome with super physiological doses of EPO, suggesting a competitive inhibition. ßCR-induced angiogenesis in HUVEC's was also abolished with treatment of P7 ßIP, but P7 ßIP did not inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis. In addition, we demonstrate that the novel P7 ßIP does not inhibit EPO-induced erythropoiesis with use of peripheral blood mononuclear cells (PBMCs). These results, for the first time, describe a novel, potent ßCR peptide inhibitor that inhibit the actions of the ßCR without affecting erythropoiesis.

Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin: Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function.

The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.

Computational modeling and in-vitro/in-silico correlation of phospholipid-based prodrugs for targeted drug delivery in inflammatory bowel disease.

Targeting drugs to the inflamed intestinal tissue(s) represents a major advancement in the treatment of inflammatory bowel disease (IBD). In this work we present a powerful in-silico modeling approach to guide the molecular design of novel prodrugs targeting the enzyme PLA2, which is overexpressed in the inflamed tissues of IBD patients. The prodrug consists of the drug moiety bound to the sn-2 position of phospholipid (PL) through a carbonic linker, aiming to allow PLA2 to release the free drug. The linker length dictates the affinity of the PL-drug conjugate to PLA2, and the optimal linker will enable maximal PLA2-mediated activation. Thermodynamic integration and Weighted Histogram Analysis Method (WHAM)/Umbrella Sampling method were used to compute the changes in PLA2 transition state binding free energy of the prodrug molecule (∆∆Gtr) associated with decreasing/increasing linker length. The simulations revealed that 6-carbons linker is the optimal one, whereas shorter or longer linkers resulted in decreased PLA2-mediated activation. These in-silico results were shown to be in excellent correlation with experimental in-vitro data. Overall, this modern computational approach enables optimization of the molecular design of novel prodrugs, which may allow targeting the free drug specifically to the diseased intestinal tissue of IBD patients.

In Silico Prediction of Ligand Binding Energies in Multiple Therapeutic Targets and Diverse Ligand Sets-A Case Study on BACE1, TYK2, HSP90, and PERK Proteins.

We present here the use of QM/MM LIE (linear interaction energy) based binding free energy calculations that greatly improve the precision and accuracy of predicting experimental binding affinities, in comparison to most current binding free energy methodologies, while maintaining reasonable computational times. Calculations are done for four sets of ligand-protein complexes, chosen on the basis of diversity of protein types and availability of experimental data, totaling 140 ligands binding to therapeutic protein targets BACE1, TYK2, HSP90, and PERK. This method allows calculations for a diverse set of ligands and multiple protein targets without the need for parametrization or extra calculations. The accuracy achieved with this method can be used to guide small molecule computational drug design programs.

Phospholipid-Based Prodrugs for Drug Targeting in Inflammatory Bowel Disease: Computational Optimization and In-Vitro Correlation.

In inflammatory bowel disease (IBD) patients, the enzyme phospholipase A2 (PLA2) is overexpressed in the inflamed intestinal tissue, and hence may be exploited as a prodrug-activating enzyme allowing drug targeting to the site(s) of gut inflammation. The purpose of this work was to develop powerful modern computational approaches, to allow optimized a-priori design of phospholipid (PL) based prodrugs for IBD drug targeting. We performed simulations that predict the activation of PL-drug conjugates by PLA2 with both human and bee venom PLA2. The calculated results correlated well with in-vitro experimental data. In conclusion, a-priori drug design using a computational approach complements and extends experimentally derived data, and may improve resource utilization and speed drug development.

Varying the electronic structure of surface-bound ruthenium(II) polypyridyl complexes.

In the design of light-harvesting chromophores for use in dye-sensitized photoelectrosynthesis cells (DSPECs), surface binding to metal oxides in aqueous solutions is often inhibited by synthetic difficulties. We report here a systematic synthesis approach for preparing a family of Ru(II) polypyridyl complexes of the type [Ru(4,4'-R2-bpy)2(4,4'-(PO3H2)2-bpy)](2+) (4,4'(PO3H2)2-bpy = [2,2'-bipyridine]-4,4'-diylbis(phosphonic acid); 4,4'-R2-bpy = 4,4'-R2-2,2'-bipyridine; and R = OCH3, CH3, H, or Br). In this series, the nature of the 4,4'-R2-bpy ligand is modified through the incorporation of electron-donating (R = OCH3 or CH3) or electron-withdrawing (R = Br) functionalities to tune redox potentials and excited-state energies. Electrochemical measurements show that the ground-state potentials, E(o')(Ru(3+/2+)), vary from 1.08 to 1.45 V (vs NHE) when the complexes are immobilized on TiO2 electrodes in aqueous HClO4 (0.1 M) as a result of increased Ru dπ-π* back-bonding caused by the lowering of the π* orbitals on the 4,4'-R2-bpy ligand. The same ligand variations cause a negligible shift in the metal-to-ligand charge-transfer absorption energies. Emission energies decrease from λmax = 644 to 708 nm across the series. Excited-state redox potentials are derived from single-mode Franck-Condon analyses of room-temperature emission spectra and are discussed in the context of DSPEC applications.

Electronic and optical properties of polypyridylruthenium derivatized polystyrenes: multi-level computational analysis of metallo-polymeric chromophore assemblies.

Great effort is geared toward investigation of new materials for solar energy conversion in recent years. Polymeric chromophore assemblies consisting of [Ru(bpy)3](2+) complexes attached to a polystyrene backbone have gained considerable interest in recent years because of their structural flexibility combined with their ability to efficiently capture solar energy and transport the captured energy in the form of exciton or charges. We employ a combination of computational methods to examine how opto-electronic properties of [Ru(bpy)3](2+) complexes are influenced by the polymer dynamics in these polymeric chromophore assemblies. The covalent linker between the polymer and the light-absorbing Ru complex is thought to play an important role in optimizing the assemblies for solar energy conversion and transport. We find that the presence of -CH2- groups in the linker has a significant impact on the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) energies of the pendants. Generally speaking, a longer linker leads to higher HOMO energies. Without the presence of -CH2- groups, a mixture of cis and trans amide bond in the covalent linker leads to a bimodal distribution for both HOMO and LUMO energies. Importantly, we find that distributions of orbital energies from individual [Ru(bpy)3](2+) pendants have the maximum overlap when there is only one -CH2- group in the linker. Such an isotropic energy distribution is likely to be important for charge transport within the assemblies. We also find that in contrast to the isolated [Ru(bpy)3](2+) complex, the HOMO is generally found on the linker rather than on Ru atom. This does not change the character of the metal-to-ligand charge transfer (MLCT) excited states, as these excitations in the pendants do not derive from HOMO/LUMO transitions but rather from HOMO - 2/LUMO transition since HOMO - 2 is located on the Ru atom.

Controlling ground and excited state properties through ligand changes in ruthenium polypyridyl complexes.

The capture and storage of solar energy requires chromophores that absorb light throughout the solar spectrum. We report here the synthesis, characterization, electrochemical, and photophysical properties of a series of Ru(II) polypyridyl complexes of the type [Ru(bpy)2(N-N)](2+) (bpy = 2,2'-bipyridine; N-N is a bidentate polypyridyl ligand). In this series, the nature of the N-N ligand was altered, either through increased conjugation or incorporation of noncoordinating heteroatoms, as a way to use ligand electronic properties to tune redox potentials, absorption spectra, emission spectra, and excited state energies and lifetimes. Electrochemical measurements show that lowering the π* orbitals on the N-N ligand results in more positive Ru(3+/2+) redox potentials and more positive first ligand-based reduction potentials. The metal-to-ligand charge transfer absorptions of all of the new complexes are mostly red-shifted compared to Ru(bpy)3(2+) (λmax = 449 nm) with the lowest energy MLCT absorption appearing at λmax = 564 nm. Emission energies decrease from λmax = 650 nm to 885 nm across the series. One-mode Franck-Condon analysis of room-temperature emission spectra are used to calculate key excited state properties, including excited state redox potentials. The impacts of ligand changes on visible light absorption, excited state reduction potentials, and Ru(3+/2+) potentials are assessed in the context of preparing low energy light absorbers for application in dye-sensitized photoelectrosynthesis cells.

Controlled electropolymerization of ruthenium(II) vinylbipyridyl complexes in mesoporous nanoparticle films of TiO(2).

Surface-initiated, oligomeric assemblies of ruthenium(II) vinylpolypyridyl complexes have been grown within the cavities of mesoporous nanoparticle films of TiO2 by electrochemically controlled radical polymerization. Surface growth was monitored by cyclic voltammetry as well as UV/Vis and X-ray photoelectron spectroscopy. Polymerization occurs by a radical chain mechanism following cyclic voltammetry scans to negative potentials where reduction occurs at the π* levels of the polypyridyl ligands. Oligomeric growth within the cavities of the TiO2 films occurs until an average of six repeat units are added to the surface-bound initiator site, which is in agreement with estimates of the internal volumes of the pores in the nanoparticle films.

Atom transfer radical polymerization preparation and photophysical properties of polypyridylruthenium derivatized polystyrenes.

A ruthenium containing polymer featuring a short carbonyl-amino-methylene linker has been prepared by atom transfer radical polymerization (ATRP). The polymer was derived from ATRP of the N-hydroxysuccinimide (NHS) derivative of p-vinylbenzoic acid, followed by an amide coupling reaction of the NHS-polystyrene with Ru(II) complexes derivatized with aminomethyl groups (i.e., [Ru(bpy)2(CH3-bpy-CH2NH2)](2+) where bpy is 2,2'-bipyridine, and CH3-bpy-CH2NH2 is 4-methyl-4'-aminomethyl-2,2'-bipyridine). The Ru-functionalized polymer structure was confirmed by using nuclear magnetic resonance and infrared spectroscopy, and the results suggest that a high loading ratio of polypyridylruthenium chromophores on the polystyrene backbone was achieved. The photophysical properties of the polymer were characterized in solution and in rigid ethylene glycol glasses. In solution, emission quantum yield and lifetime studies reveal that the polymer's metal-to-ligand charge transfer (MLCT) excited states are quenched relative to a model Ru complex chromophore. In rigid media, the MLCT-ground state band gap and lifetime are both increased relative to solution with time-resolved emission measurements revealing fast energy transfer hopping within the polymer. Molecular dynamics studies of the polymer synthesized here as well as similar model systems with various spatial arrangements of the pendant Ru complex chromophores suggest that the carbonyl-amino-methylene linker probed in our target polymer provides shorter Ru-Ru nearest-neighbor distances leading to an increased Ru*-Ru energy hopping rate, compared to those with longer linkers in counterpart polymers.

Interfacial hydration, dynamics and electron transfer: multi-scale ET modeling of the transient [myoglobin, cytochrome b5] complex.

Formation of a transient [myoglobin (Mb), cytochrome b(5) (cyt b(5))] complex is required for the reductive repair of inactive ferri-Mb to its functional ferro-Mb state. The [Mb, cyt b(5)] complex exhibits dynamic docking (DD), with its cyt b(5) partner in rapid exchange at multiple sites on the Mb surface. A triple mutant (Mb(3M)) was designed as part of efforts to shift the electron-transfer process to the simple docking (SD) regime, in which reactive binding occurs at a restricted, reactive region on the Mb surface that dominates the docked ensemble. An electrostatically-guided brownian dynamics (BD) docking protocol was used to generate an initial ensemble of reactive configurations of the complex between unrelaxed partners. This ensemble samples a broad and diverse array of heme-heme distances and orientations. These configurations seeded all-atom constrained molecular dynamics simulations (MD) to generate relaxed complexes for the calculation of electron tunneling matrix elements (T(DA)) through tunneling-pathway analysis. This procedure for generating an ensemble of relaxed complexes combines the ability of BD calculations to sample the large variety of available conformations and interprotein distances, with the ability of MD to generate the atomic level information, especially regarding the structure of water molecules at the protein-protein interface, that defines electron-tunneling pathways. We used the calculated T(DA) values to compute ET rates for the [Mb(wt), cyt b(5)] complex and for the complex with a mutant that has a binding free energy strengthened by three D/E → K charge-reversal mutations, [Mb(3M), cyt b(5)]. The calculated rate constants are in agreement with the measured values, and the mutant complex ensemble has many more geometries with higher T(DA) values than does the wild-type Mb complex. Interestingly, water plays a double role in this electron-transfer system, lowering the tunneling barrier as well as inducing protein interface remodeling that screens the repulsion between the negatively-charged propionates of the two hemes.

Effect of backbone flexibility on charge transfer rates in peptide nucleic acid duplexes.

Charge transfer (CT) properties are compared between peptide nucleic acid structures with an aminoethylglycine backbone (aeg-PNA) and those with a γ-methylated backbone (γ-PNA). The common aeg-PNA is an achiral molecule with a flexible structure, whereas γ-PNA is a chiral molecule with a significantly more rigid structure than aeg-PNA. Electrochemical measurements show that the CT rate constant through an aeg-PNA bridging unit is twice the CT rate constant through a γ-PNA bridging unit. Theoretical calculations of PNA electronic properties, which are based on a molecular dynamics structural ensemble, reveal that the difference in the CT rate constant results from the difference in the extent of backbone fluctuations of aeg- and γ-PNA. In particular, fluctuations of the backbone affect the local electric field that broadens the energy levels of the PNA nucleobases. The greater flexibility of the aeg-PNA gives rise to more broadening, and a more frequent appearance of high-CT rate conformations than in γ-PNA.

Nucleic Acid Charge Transfer: Black, White and Gray.

Theoretical studies of charge transport in deoxyribonucleic acid (DNA) and peptide nucleic acid (PNA) indicate that structure and dynamics modulate the charge transfer rates, and that different members of a structural ensemble support different charge transport mechanisms. Here, we review the influences of nucleobase geometry, electronic structure, solvent environment, and thermal conformational fluctuations on the charge transfer mechanism. We describe an emerging framework for understanding the diversity of charge transport mechanisms seen in nucleic acids.