A Pd-Catalyzed Annulation Strategy to Linearly Fused Functionalized N-Heterocycles.

Linearly fused polycyclic piperidines represent common substructures in natural products and biologically active small molecules. We have devised a Pd-catalyzed annulation strategy to these compounds that converts readily available 2-tetralones and indanones into these scaffolds with the potential for control of both enantio- and diastereoselectivity. Importantly, these compounds can be chemoselectively functionalized, providing an efficient and robust methodology to these important nitrogen-containing molecules.

Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical.

All cells obtain 2'-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, ß; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in ß. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the ß is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.

Evidence for Modulation of Oxygen Rebound Rate in Control of Outcome by Iron(II)- and 2-Oxoglutarate-Dependent Oxygenases.

Iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases generate iron(IV)-oxo (ferryl) intermediates that can abstract hydrogen from aliphatic carbons (R-H). Hydroxylation proceeds by coupling of the resultant substrate radical (R•) and oxygen of the Fe(III)-OH complex ("oxygen rebound"). Nonhydroxylation outcomes result from different fates of the Fe(III)-OH/R• state; for example, halogenation results from R• coupling to a halogen ligand cis to the hydroxide. We previously suggested that halogenases control substrate-cofactor disposition to disfavor oxygen rebound and permit halogen coupling to prevail. Here, we explored the general implication that, when a ferryl intermediate can ambiguously target two substrate carbons for different outcomes, rebound to the site capable of the alternative outcome should be slower than to the adjacent, solely hydroxylated site. We evaluated this prediction for (i) the halogenase SyrB2, which exclusively hydroxylates C5 of norvaline appended to its carrier protein but can either chlorinate or hydroxylate C4 and (ii) two bifunctional enzymes that normally hydroxylate one carbon before coupling that oxygen to a second carbon (producing an oxacycle) but can, upon encountering deuterium at the first site, hydroxylate the second site instead. In all three cases, substrate hydroxylation incorporates a greater fraction of solvent-derived oxygen at the site that can also undergo the alternative outcome than at the other site, most likely reflecting an increased exchange of the initially O2-derived oxygen ligand in the longer-lived Fe(III)-OH/R• states. Suppression of rebound may thus be generally important for nonhydroxylation outcomes by these enzymes.

Hydrogen Donation but not Abstraction by a Tyrosine (Y68) during Endoperoxide Installation by Verruculogen Synthase (FtmOx1).

Hydrogen-atom transfer (HAT) from a substrate carbon to an iron(IV)-oxo (ferryl) intermediate initiates a diverse array of enzymatic transformations. For outcomes other than hydroxylation, coupling of the resultant carbon radical and hydroxo ligand (oxygen rebound) must generally be averted. A recent study of FtmOx1, a fungal iron(II)- and 2-(oxo)glutarate-dependent oxygenase that installs the endoperoxide of verruculogen by adding O2 between carbons 21 and 27 of fumitremorgin B, posited that tyrosine (Tyr or Y) 224 serves as HAT intermediary to separate the C21 radical (C21•) and Fe(III)-OH HAT products and prevent rebound. Our reinvestigation of the FtmOx1 mechanism revealed, instead, direct HAT from C21 to the ferryl complex and surprisingly competitive rebound. The C21-hydroxylated (rebound) product, which undergoes deprenylation, predominates when low [O2] slows C21•-O2 coupling in the next step of the endoperoxidation pathway. This pathway culminates with addition of the C21-O-O• peroxyl adduct to olefinic C27 followed by HAT to the C26• from a Tyr. The last step results in sequential accumulation of Tyr radicals, which are suppressed without detriment to turnover by inclusion of the reductant, ascorbate. Replacement of each of four candidates for the proximal C26 H• donor (including Y224) with phenylalanine (F) revealed that only the Y68F variant (i) fails to accumulate the first Tyr• and (ii) makes an altered major product, identifying Y68 as the donor. The implied proximities of C21 to the iron cofactor and C26 to Y68 support a new docking model of the enzyme-substrate complex that is consistent with all available data.

Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins.

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility.

Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase.

Iron(II)- and 2-(oxo)-glutarate-dependent oxygenases catalyze diverse oxidative transformations that are often initiated by abstraction of hydrogen from carbon by iron(IV)-oxo (ferryl) complexes. Control of the relative orientation of the substrate C-H and ferryl Fe-O bonds, primarily by direction of the oxo group into one of two cis-related coordination sites (termed inline and offline), may be generally important for control of the reaction outcome. Neither the ferryl complexes nor their fleeting precursors have been crystallographically characterized, hindering direct experimental validation of the offline hypothesis and elucidation of the means by which the protein might dictate an alternative oxo position. Comparison of high-resolution X-ray crystal structures of the substrate complex, an Fe(II)-peroxysuccinate ferryl precursor, and a vanadium(IV)-oxo mimic of the ferryl intermediate in the l-arginine 3-hydroxylase, VioC, reveals coordinated motions of active site residues that appear to control the intermediate geometries to determine reaction outcome.

Utilizing Palladium-Stabilized Zwitterions for the Construction of N-Heterocycles.

Pd catalysis provides a convenient method for the generation of zwitterionic intermediates that offer significant opportunities for the synthesis of functionalized heterocycles. The combination of electrophilic π-allyl Pd fragments and C-, N- and O-centered nucleophiles allows these intermediates to react with readily available substrates to furnish a range of high value products with control of chemo-, regio- and stereo-selectivity.

A Pd-Catalyzed Synthesis of Functionalized Piperidines.

A readily available cyclic carbamate 1 functions as a general precursor to a range of functionalized piperidine products via a new Pd-catalyzed annulation strategy. An asymmetric catalytic variant provides a rapid and efficient means to access these heterocycles with high to excellent levels of enantiocontrol. Finally, these richly functionalized compounds are amenable to further chemoselective elaboration.

Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles.

The stability, activity, and solubility of a protein sequence are determined by a delicate balance of molecular interactions in a variety of conformational states. Even so, most computational protein design methods model sequences in the context of a single native conformation. Simulations that model the native state as an ensemble have been mostly neglected due to the lack of sufficiently powerful optimization algorithms for multistate design. Here, we have applied our multistate design algorithm to study the potential utility of various forms of input structural data for design. To facilitate a more thorough analysis, we developed new methods for the design and high-throughput stability determination of combinatorial mutation libraries based on protein design calculations. The application of these methods to the core design of a small model system produced many variants with improved thermodynamic stability and showed that multistate design methods can be readily applied to large structural ensembles. We found that exhaustive screening of our designed libraries helped to clarify several sources of simulation error that would have otherwise been difficult to ascertain. Interestingly, the lack of correlation between our simulated and experimentally measured stability values shows clearly that a design procedure need not reproduce experimental data exactly to achieve success. This surprising result suggests potentially fruitful directions for the improvement of computational protein design technology.

Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries.

The longer emission wavelengths of red fluorescent proteins (RFPs) make them attractive for whole-animal imaging because cells are more transparent to red light. Although several useful RFPs have been developed using directed evolution, the quest for further red-shifted and improved RFPs continues. Herein, we report a structure-based rational design approach to red-shift the fluorescence emission of RFPs. We applied a combined computational and experimental approach that uses computational protein design as an in silico prescreen to generate focused combinatorial libraries of mCherry mutants. The computational procedure helped us identify residues that could fulfill interactions hypothesized to cause red-shifts without destabilizing the protein fold. These interactions include stabilization of the excited state through H-bonding to the acylimine oxygen atom, destabilization of the ground state by hydrophobic packing around the charged phenolate, and stabilization of the excited state by a π-stacking interaction. Our methodology allowed us to identify three mCherry mutants (mRojoA, mRojoB, and mRouge) that display emission wavelengths > 630 nm, representing red-shifts of 20-26 nm. Moreover, our approach required the experimental screening of a total of ∼5,000 clones, a number several orders of magnitude smaller than those previously used to achieve comparable red-shifts. Additionally, crystal structures of mRojoA and mRouge allowed us to verify fulfillment of the interactions hypothesized to cause red-shifts, supporting their contribution to the observed red-shifts.

An efficient algorithm for multistate protein design based on FASTER.

Most of the methods that have been developed for computational protein design involve the selection of side-chain conformations in the context of a single, fixed main-chain structure. In contrast, multistate design (MSD) methods allow sequence selection to be driven by the energetic contributions of multiple structural or chemical states simultaneously. This methodology is expected to be useful when the design target is an ensemble of related states rather than a single structure, or when a protein sequence must assume several distinct conformations to function. MSD can also be used with explicit negative design to suggest sequences with altered structural, binding, or catalytic specificity. We report implementation details of an efficient multistate design optimization algorithm based on FASTER (MSD-FASTER). We subjected the algorithm to a battery of computational tests and found it to be generally applicable to various multistate design problems; designs with a large number of states and many designed positions are completely feasible. A direct comparison of MSD-FASTER and multistate design Monte Carlo indicated that MSD-FASTER discovers low-energy sequences much more consistently. MSD-FASTER likely performs better because amino acid substitutions are chosen on an energetic basis rather than randomly, and because multiple substitutions are applied together. Through its greater efficiency, MSD-FASTER should allow protein designers to test experimentally better-scoring sequences, and thus accelerate progress in the development of improved scoring functions and models for computational protein design.

Self-Assembly of Topologically Networked Protein-Ti3C2Tx MXene Composites.

Hierarchical organization plays an important role in the stunning physical properties of natural and synthetic composites. Limits on the physical properties of such composites are generally defined by percolation theory and can be systematically altered using the volumetric filler fraction of the inorganic/organic phase. In natural composites, organic materials such as proteins that interact with inorganic filler materials can further alter the hierarchical order and organization of the composite via topological interactions, expanding the limits of the physical properties defined by percolation theory. However, existing polymer systems do not offer a topological parameter that can systematically modulate the assembly characteristics of composites. Here, we present a composite based on proteins and titanium carbide (Ti3C2Tx) MXene that manifests a topological network that regulates the organization, and hence physical properties, of these biomimetic composites. We designed, recombinantly expressed, and purified synthetic proteins consisting of polypeptides with repeating amino acid sequences (tandem repeats) that have the ability to self-assemble into topologically networked biomaterials. We demonstrated that the interlayer distance between MXene sheets can be controlled systematically by the number of tandem repeat units. We varied the filler fraction and number of tandem repeat units to regulate the in-plane and out-of-plane electrical conductivities of these composites. Once Ti3C2Tx MXene sheets are separated enough to facilitate formation of cross-links in our proteins with the number of tandem repeat units reaching 11, the linear I-V characteristics of the composites switched into nonlinear I-V curves with a distinct hysteresis for out-of-plane electron transport, while the in-plane I-V characteristics remained linear. This highlights the impact of synthetic protein templates, which can be designed to modulate electronic transport in composites both isotropically and anisotropically.

Manganese-Catalyzed Electrochemical Deconstructive Chlorination of Cycloalkanols via Alkoxy Radicals.

A manganese-catalyzed electrochemical deconstructive chlorination of cycloalkanols has been developed. This electrochemical method provides access to alkoxy radicals from alcohols and exhibits a broad substrate scope, with various cyclopropanols and cyclobutanols converted into synthetically useful ß- and γ-chlorinated ketones (40 examples). Furthermore, the combination of recirculating flow electrochemistry and continuous inline purification was employed to access products on a gram scale.

Conformational analysis of a covalently cross-linked Watson-Crick base pair model.

Low-temperature NMR experiments and molecular modeling have been used to characterize the conformational behavior of a covalently cross-linked DNA base pair model. The data suggest that Watson-Crick or reverse Watson-Crick hydrogen bonding geometries have similar energies and can interconvert at low temperatures. This low-temperature process involves rotation about the crosslink CH(2)C(5') (psi) carbon-carbon bond, which is energetically preferred over the alternate CH(2)N(3) (phi) carbon-nitrogen bond rotation.

Mechanical Properties of Tandem-Repeat Proteins Are Governed by Network Defects.

Topological defects in highly repetitive structural proteins strongly affect their mechanical properties. However, there are no universal rules for structure-property prediction in structural proteins due to high diversity in their repetitive modules. Here, we studied the mechanical properties of tandem-repeat proteins inspired by squid ring teeth proteins using rheology and tensile experiments as well as spectroscopic and X-ray techniques. We also developed a network model based on entropic elasticity to predict structure-property relationships for these proteins. We demonstrated that shear modulus, elastic modulus, and toughness scale inversely with the number of repeats in these proteins. Through optimization of structural repeats, we obtained highly efficient protein network topologies with 42 MJ/m3 ultimate toughness that are capable of withstanding deformations up to 350% when hydrated. Investigation of topological network defects in structural proteins will improve the prediction of mechanical properties for designing novel protein-based materials.

Tunable thermal transport and reversible thermal conductivity switching in topologically networked bio-inspired materials.

The dynamic control of thermal transport properties in solids must contend with the fact that phonons are inherently broadband. Thus, efforts to create reversible thermal conductivity switches have resulted in only modest on/off ratios, since only a relatively narrow portion of the phononic spectrum is impacted. Here, we report on the ability to modulate the thermal conductivity of topologically networked materials by nearly a factor of four following hydration, through manipulation of the displacement amplitude of atomic vibrations. By varying the network topology, or crosslinked structure, of squid ring teeth-based bio-polymers through tandem-repetition of DNA sequences, we show that this thermal switching ratio can be directly programmed. This on/off ratio in thermal conductivity switching is over a factor of three larger than the current state-of-the-art thermal switch, offering the possibility of engineering thermally conductive biological materials with dynamic responsivity to heat.

ProtaBank: A repository for protein design and engineering data.

We present ProtaBank, a repository for storing, querying, analyzing, and sharing protein design and engineering data in an actively maintained and updated database. ProtaBank provides a format to describe and compare all types of protein mutational data, spanning a wide range of properties and techniques. It features a user-friendly web interface and programming layer that streamlines data deposition and allows for batch input and queries. The database schema design incorporates a standard format for reporting protein sequences and experimental data that facilitates comparison of results across different data sets. A suite of analysis and visualization tools are provided to facilitate discovery, to guide future designs, and to benchmark and train new predictive tools and algorithms. ProtaBank will provide a valuable resource to the protein engineering community by storing and safeguarding newly generated data, allowing for fast searching and identification of relevant data from the existing literature, and exploring correlations between disparate data sets. ProtaBank invites researchers to contribute data to the database to make it accessible for search and analysis. ProtaBank is available at https://protabank.org.

Computational protein design promises to revolutionize protein engineering.

Natural evolution has produced an astounding array of proteins that perform the physical and chemical functions required for life on Earth. Although proteins can be reengineered to provide altered or novel functions, the utility of this approach is limited by the difficulty of identifying protein sequences that display the desired properties. Recently, advances in the field of computational protein design (CPD) have shown that molecular simulation can help to predict sequences with new and improved functions. In the past few years, CPD has been used to design protein variants with optimized specificity of binding to DNA, small molecules, peptides, and other proteins. Initial successes in enzyme design highlight CPD's unique ability to design function de novo. The use of CPD for the engineering of potential therapeutic agents has demonstrated its strength in real-life applications.