Departmental Papers (Chemistry)

Penn Chemistry is a leading center of molecular research. Within the department, the traditional sub-fields of biochemistry, organic, inorganic, and physical chemistry serve as a framework from which collaborative, inter-disciplinary experimental and theoretical research is launched. Research thrusts include:

  • Synthesis and Characterization of New Compounds and Materials
  • Chemistry of Life Processes
  • Nanoscale Materials and Molecular Devices
  • Chemistry of the Environment
  • Energy and the Hydrogen Economy

 

 

Department of Chemistry

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  • Publication
    Computational Design of Water-Soluble Analogues of the Potassium Channel KcsA
    (2004-02-17) Lear, James D; Slovic, Avram Michael; Saven, Jeffery G.; Kono, Hidetoshi; DeGrado, William F
    Although the interiors of membrane and water-soluble proteins are similar in their physicochemical properties, membrane proteins differ in having larger fractions of hydrophobic residues on their exteriors. Thus, it should be possible to water-solubilize membrane proteins by mutating their lipid-contacting side chains to more polar groups. Here, a computational approach was used to generate water-soluble variants of the potassium channel KcsA. As a probe of the correctness of the fold, the proteins contain an agitoxin2 binding site from a mammalian homologue of the channel. The resulting proteins express in high yield inEscherichia coli and share the intended functional and structural properties with KcsA, including secondary structure, tetrameric quaternary structure, and tight specific binding to both agitoxin2 and a small molecule channel blocker.
  • Publication
    NMR Structure and Dynamics of a Designed Water-Soluble Transmembrane Domain of Nicotinic Acetylcholine Receptor
    (2012-03-01) Cui, Tanxing; Mowrey, David; Bondarenko, Vasyl; Tillman, Tommy; Ma, Dejian; Perez Aguilar, Jose Manuel; Landrum, Elizabeth; He, Jing; Saven, Jeffery G.; Wang, Wei; Eckenhoff, Roderic G; Tang, Pei; Xu, Yan
    The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828–1833, 2004; Ma et al., PNAS, 105: 16537–42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~ 83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceuspentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T2 dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.
  • 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 F
    We 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
    Knowledge-Based Potential for Positioning Membrane-Associated Structures and Assessing Residue-Specific Energetic Contributions
    (2012-05-09) Hannigan, Brett Thomas; Schramm, Chaim A; Saven, Jeffery G.; Donald, Jason E; DeGrado, William F; Keasar, Chen; Samish, Ilan
    The complex hydrophobic and hydrophilic milieus of membrane-associated proteins pose experimental and theoretical challenges to their understanding. Here we produce a non-redundant database to compute knowledge-based asymmetric cross-membrane potentials from the per-residue distributions of Cβ, Cγ and functional group atoms. We predict transmembrane and peripherally associated regions from genomic sequence and position peptides and protein structures relative to the bilayer (available at http://www.degradolab.org/ez). The pseudo-energy topological landscapes underscore positional stability and functional mechanisms demonstrated here for antimicrobial peptides, transmembrane proteins, and viral fusion proteins. Moreover, experimental effects of point mutations on the relative ratio changes of dual-topology proteins are quantitatively reproduced. The functional group potential and the membrane-exposed residues display the largest energetic changes enabling to detect native-like structures from decoys. Hence, focusing on the uniqueness of membrane-associated proteins and peptides, we quantitatively parameterize their cross-membrane propensity thus facilitating structural refinement, characterization, prediction and design.
  • Publication
    Computational Design of Membrane Proteins
    (2012-01-11) Perez Aguilar, Jose Manuel; Saven, Jeffery G.
    Membrane proteins are involved in a wide variety of cellular processes, and are typically part of the first interaction a cell has with extracellular molecules. As a result, these proteins comprise a majority of known drug targets. Membrane proteins are among the most difficult proteins to obtain and characterize, and a structure-based understanding of their properties can be difficult to elucidate. Notwithstanding, the design of membrane proteins can provide stringent tests of our understanding of these crucial biological systems, as well as introduce novel or targeted functionalities. Computational design methods have been particularly helpful in addressing these issues and this review discusses recent studies that tailor membrane proteins to display specific structures or functions, and how redesigned membrane proteins are being used to facilitate structural and functional studies.
  • 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
    Computational Design of a Protein Crystal
    (2012-05-08) Lanci, Christopher J; MacDermaid, Christopher M; Keng, Seung-gu; Acharya, Rudresh; North, Benjamin; Yang, Xi; DeGrado, William F; Qiu, X Jade; Saven, Jeffery G.
    Protein crystals have catalytic and materials applications and are central to efforts in structural biology and therapeutic development. Designing predetermined crystal structures can be subtle given the complexity of proteins and the noncovalent interactions that govern crystallization. De novo protein design provides an approach to engineer highly complex nanoscale molecular structures, and often the positions of atoms can be programmed with sub-Å precision. Herein, a computational approach is presented for the design of proteins that self-assemble in three dimensions to yield macroscopic crystals. A three-helix coiled-coil protein is designed de novo to form a polar, layered, three-dimensional crystal having the P6 space group, which has a “honeycomb-like” structure and hexameric channels that span the crystal. The approach involves: (i) creating an ensemble of crystalline structures consistent with the targeted symmetry; (ii) characterizing this ensemble to identify “designable” structures from minima in the sequence-structure energy landscape and designing sequences for these structures; (iii) experimentally characterizing candidate proteins. A 2.1 Å resolution X-ray crystal structure of one such designed protein exhibits sub-Å agreement [backbone root mean square deviation (rmsd)] with the computational model of the crystal. This approach to crystal design has potential applications to the de novo design of nanostructured materials and to the modification of natural proteins to facilitate X-ray crystallographic analysis.
  • Publication
    Computational Protein Design to Re-Engineer Stromal Cell-Derived Factor-1α (SDF) Generates an Effective and Translatable Angiogenic Polypeptide Analog
    (2011-09-13) Perez Aguilar, Jose Manuel; Hiesinger, William; Atluri, Pavan; Marotta, Nicole A; Frederick, John R; Fitzpatrick, J Raymond; McCormick, Ryan C; Muenzer, Jeffrey R; Yang, Elaine C; Levit, Rebecca D; Yuan, Li-Jun; Saven, Jeffery G.; Macarthur, John W; Woo, Y Joseph
    BACKGROUND: Experimentally, exogenous administration of recombinant stromal cell-derived factor-1α (SDF) enhances neovasculogenesis and cardiac function after myocardial infarction. Smaller analogs of SDF may provide translational advantages including enhanced stability and function, ease of synthesis, lower cost, and potential modulated delivery via engineered biomaterials. In this study, computational protein design was used to create a more efficient evolution of the native SDF protein. METHODS AND RESULTS: Protein structure modeling was used to engineer an SDF polypeptide analog (engineered SDF analog [ESA]) that splices the N-terminus (activation and binding) and C-terminus (extracellular stabilization) with a diproline segment designed to limit the conformational flexibility of the peptide backbone and retain the relative orientation of these segments observed in the native structure of SDF. Endothelial progenitor cells (EPCs) in ESA gradient, assayed by Boyden chamber, showed significantly increased migration compared with both SDF and control gradients. EPC receptor activation was evaluated by quantification of phosphorylated AKT, and cells treated with ESA yielded significantly greater phosphorylated AKT levels than SDF and control cells. Angiogenic growth factor assays revealed a distinct increase in angiopoietin-1 expression in the ESA- and SDF-treated hearts. In addition, CD-1 mice (n=30) underwent ligation of the left anterior descending coronary artery and peri-infarct intramyocardial injection of ESA, SDF-1α, or saline. At 2 weeks, echocardiography demonstrated a significant gain in ejection fraction, cardiac output, stroke volume, and fractional area change in mice treated with ESA compared with controls. CONCLUSIONS: Compared with native SDF, a novel engineered SDF polypeptide analog (ESA) more efficiently induces EPC migration and improves post-myocardial infarction cardiac function and thus offers a more clinically translatable neovasculogenic therapy.
  • 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 J
    The 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
    Characterization of a Computationally Designed Water-Soluble Human μ Opioid Receptor Variant Using X-ray Structural Information
    (2014-10-01) Perez Aguilar, Jose Manuel; Zhao, Xuelian; Matsunaga, Felipe; Lerner, Mitchell Bryant; Xi, Jin; Selling, Bernard; Saven, Jeffery G.; Johnson, A. T. Charlie; Liu, Renyu
    Background The recent X-ray crystal structure of the murine μ opioid receptor (MUR) allowed us to reengineer a previously designed water-soluble variant of the transmembrane portion of the human MUR (wsMUR-TM). Methods The new variant of water soluble MUR (wsMUR-TM_v2) was engineered based upon the murine MUR crystal structure. This novel variant was expressed in E. coliand purified. The properties of the receptor were characterized and compared with those of wsMUR-TM. Results Seven residues originally included for mutation in the design of the wsMUR-TM, were reverted to their native identities. wsMUR-TM_v2 contains 16% mutations of the total sequence. It was overexpressed and purified with high yield. Although dimers and higher oligomers were observed to form over time, the wsMUR-TM_v2 stayed predominantly monomeric at concentrations as high as 7.5 mg/ml in buffer within a 2-month period. Its secondary structure was predominantly helical and comparable with those of both the original wsMUR-TM variant and the native MUR. The binding affinity of wsMUR-TM_v2 for naltrexone (Kd ~ 70 nM) was in close agreement with that for wsMUR-TM. The helical content of wsMUR-TM_v2 decreased cooperatively with increasing temperature, and the introduction of sucrose was able to stabilize the protein. Conclusions A novel functional wsMUR-TM_v2 with only 16% mutations was successfully engineered, expressed in E. coli and purified based on information from the crystal structure of murine MUR. This not only provides a novel alternative tool for MUR studies in solution conditions, but also offers valuable information for protein engineering and structure function relationships.