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Publication Computational Design and Elaboration of a De Novo Heterotetrameric α-Helical Protein that Selectively Binds an Emissive Abiological (Porphinato)zinc Chromophore(2010-03-24) Saven, Jeffery G.; Fry, H Christopher; DeGrado, William F; Lehmann, Andreas; Therien, Michael JThe first example of a computationally de novo designed protein that binds an emissive abiological chromophore is presented, in which a sophisticated level of cofactor discrimination is pre-engineered. This heterotetrameric, C(2)-symmetric bundle, A(His):B(Thr), uniquely binds (5,15-di[(4-carboxymethyleneoxy)phenyl]porphinato)zinc [(DPP)Zn] via histidine coordination and complementary noncovalent interactions. The A(2)B(2) heterotetrameric protein reflects ligand-directed elements of both positive and negative design, including hydrogen bonds to second-shell ligands. Experimental support for the appropriate formulation of [(DPP)Zn:A(His):B(Thr)](2) is provided by UV/visible and circular dichroism spectroscopies, size exclusion chromatography, and analytical ultracentrifugation. Time-resolved transient absorption and fluorescence spectroscopic data reveal classic excited-state singlet and triplet PZn photophysics for the A(His):B(Thr):(DPP)Zn protein (k(fluorescence) = 4 x 10(8) s(-1); tau(triplet) = 5 ms). The A(2)B(2) apoprotein has immeasurably low binding affinities for related [porphinato]metal chromophores that include a (DPP)Fe(III) cofactor and the zinc metal ion hemin derivative [(PPIX)Zn], underscoring the exquisite active-site binding discrimination realized in this computationally designed protein. Importantly, elements of design in the A(His):B(Thr) protein ensure that interactions within the tetra-alpha-helical bundle are such that only the heterotetramer is stable in solution; corresponding homomeric bundles present unfavorable ligand-binding environments and thus preclude protein structural rearrangements that could lead to binding of (porphinato)iron cofactors.Publication Computational Protein Design: Engineering Molecular Diversity, Nonnatural Enzymes, Nonbiological Cofactor Complexes, and Membrane Proteins(2011-06-01) Saven, Jeffery G.Computational and theoretical methods are advancing protein design as a means to create and investigate proteins. Such efforts further our capacity to control, design and understand biomolecular structure, sequence and function. Herein, the focus is on some recent applications that involve using theoretical and computational methods to guide the design of protein sequence ensembles, new enzymes, proteins with novel cofactors, and membrane proteins.Publication Using α-Helical Coiled-Coils to Design Nanostructured Metalloporphyrin Arrays(2008-09-10) McAllister, Karen A; Zou, Hongling; Cochran, Frank V; Bender, Gretchen M; Senes, Alessandro; Fry, H Christopher; Nanda, Vikas; Saven, Jeffery G.; Keenan, Patricia A; Therien, Michael J; Lear, James D; DeGrado, William F; Blasie, J KentWe have developed a computational design strategy based on the alpha-helical coiled-coil to generate modular peptide motifs capable of assembling into metalloporphyrin arrays of varying lengths. The current study highlights the extension of a two-metalloporphyrin array to a four-metalloporphyrin array through the incorporation of a coiled-coil repeat unit. Molecular dynamics simulations demonstrate that the initial design evolves rapidly to a stable structure with a small rmsd compared to the original model. Biophysical characterization reveals elongated proteins of the desired length, correct cofactor stoichiometry, and cofactor specificity. The successful extension of the two-porphyrin array demonstrates how this methodology serves as a foundation to create linear assemblies of organized electrically and optically responsive cofactors.Publication De Novo Design of a Single Chain Diphenylporphyrin Metalloprotein(2007-09-05) Bender, Gretchen M; Lehmann, Andreas; Zou, Hongling; Cheng, Hong; Fry, H Christopher; Engel, Don; Therien, Michael J; Blasie, J Kent; Saven, Jeffery G.; Roder, Heinrich; DeGrado, William FWe describe the computational design of a single-chain four-helix bundle that noncovalently self-assembles with fully synthetic non-natural porphyrin cofactors. With this strategy, both the electronic structure of the cofactor as well as its protein environment may be varied to explore and modulate the functional and photophysical properties of the assembly. Solution characterization (NMR, UV-vis) of the protein showed that it bound with high specificity to the desired cofactors, suggesting that a uniquely structured protein and well-defined site had indeed been created. This provides a genetically expressed single-chain protein scaffold that will allow highly facile, flexible, and asymmetric variations to enable selective incorporation of different cofactors, surface-immobilization, and introduction of spectroscopic probes.Publication Computational Protein Design: Advances in the Design and Redesign of Biomolecular Nanostructures(2010-04-01) Saven, Jeffery G.Computational protein design facilitates the continued development of methods for the design of biomolecular structure, sequence and function. Recent applications include the design of novel protein sequences and structures, proteins incorporating nonbiological components, protein assemblies, soluble variants of membrane proteins, and proteins that modulate membrane function.Publication Characterization of Cofactor-Induced Folding Mechanism of a Zinc Binding Peptide Using Computationally Designed Mutants(2009-05-29) Tang, Jia; Kang, Seung-Gu; Saven, Jeffery G.; Gai, FengMetals are the most commonly encountered protein cofactors, and they play important structural and functional roles in biology. In many cases, metal binding provides a major driving force for a polypeptide chain to fold. While there are many studies on the structure, stability, and function of metal-binding proteins, there are few studies focusing on understanding the kinetic mechanism of metal-induced folding. Herein, the Zn(2+)-induced folding kinetics of a small zinc-binding protein are studied; the CH1(1) peptide is derived from the first cysteine/histidine-rich region (CH1 domain) of the protein interaction domains of the transcriptional coregulator CREB-binding protein. Computational design is used to introduce tryptophan and histidine mutations that are structurally consistent with CH1(1); these mutants are studied using stopped-flow tryptophan fluorescence experiments. The Zn(2+)-induced CH1(1) folding kinetics are consistent with two parallel pathways, where the initial binding of Zn(2+) occurs at two sites. However, the initially formed Zn(2+)-bound complexes can proceed either directly to the folded state where zinc adopts a tetrahedral coordination or to an off-pathway misligated intermediate. While elimination of those ligands responsible for misligation simplifies the folding kinetics, it also leads to a decrease in the zinc binding constant. Therefore, these results suggest why these nonnative zinc ligands in the CH1(1) motif are conserved in several distantly related organisms and why the requirement for function can lead to kinetic frustration in folding. In addition, the loop closure rate of the CH1(1) peptide is determined based on the proposed model and temperature-dependent kinetic measurements.Publication Fluorescence Correlation Spectroscopic Study of Serpin Depolymerization by Computationally Designed Peptides(2007-06-01) Chowdhury, Pramit; Wang, Wei; Lavender, Stacey; Bunagan, Michelle R; Klemke, Jason W; Tang, Jia; Saven, Jeffery G.; Cooperman, Barry S; Gai, FengMembers of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within beta-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the beta-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within beta-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of alpha(1)-antitrypsin (alpha(1)-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found.Publication Xe Affinities of Water-Soluble Cryptophanes and the Role of Confined Water(2015-12-01) Gao, Lu; Liu, Wenhao; Lee, One-Sun; Dmochowskia, Ivan J; Saven, Jeffery G.Given their relevance to drug design and chemical sensing, host–guest interactions are of broad interest in molecular science. Natural and synthetic host molecules provide vehicles for understanding selective molecular recognition in aqueous solution. Here, cryptophane–Xe host–guest systems are considered in aqueous media as a model molecular system that also has important applications. 129Xe–cryptophane systems can be used in the creation of biosensors and powerful contrast agents for magnetic resonance imaging applications. Detailed molecular information on the determinants of Xe affinity is difficult to obtain experimentally. Thus, molecular simulation and free energy perturbation methods were applied to estimate the affinities of Xe for six water-soluble cryptophanes. The calculated affinities correlated well with the previously measured experimental values. The simulations provided molecular insight on the differences in affinities and the roles of conformational fluctuations, solvent, and counter ions on Xe binding to these host molecules. Displacement of confined water from the host interior cavity is a key component of the binding equilibrium, and the average number of water molecules within the host cavity is correlated with the free energy of Xe binding to the different cryptophanes. The findings highlight roles for molecular simulation and design in modulating the relative strengths of host–guest and host–solvent interactions.Publication The Role of Dynamic Ligand Exchange in the Oxidation Chemistry of Cerium(III)(2016-03-22) Robinson, Jerome R; Qiao, Yusen; Gu, Jun; Walsh, Patrick J; Carroll, Patrick J; Schelter, Eric JThe CeIII/IV couple is useful for many applications in organic, inorganic, and materials chemistry. However, attaining a general method to access both oxidations states through reversible solution redox chemistry remains challenging. Herein we report the synthesis, characterization, and oxidation chemistry of the novel Ce/Li REMB heterochiral diastereomer, 1-Ce(het). The solution exchange processes of 1-RE(het) (RE ¼ Ce and Yb) were investigated to estimate rates of ligand and cation exchange relevant in homochiral and heterochiral frameworks. A detailed mechanistic investigation following the solution dynamics of 1-Ce(het) revealed reactivity controlled both by ligand reorganization and redistribution processes. Ligand reorganization was responsible for the kinetics associated with the chemical oxidation reaction, whereas ligand redistribution and exchange dictated the isolated productsPublication A Computationally Designed Water-Soluble Variant of a G-Protein-Coupled Receptor: The Human Mu Opioid Receptor(2013-06-14) Perez Aguilar, Jose Manuel; Xi, Jin; Matsunaga, Felipe; Cui, Xu; Saven, Jeffery G.; Selling, Bernard; Liu, RenyuG-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (Kd = 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.
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