Saven, Jeffery G.
Email Address
ORCID
Disciplines
relationships.isProjectOf
relationships.isOrgUnitOf
Position
Introduction
Research Interests
Collection
26 results
Search Results
Now showing 1 - 10 of 26
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 Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation(2009-08-01) Griffon, Nathalie; Petty II, Thomas John; Jin, Weijin; Millar, John; Saven, Jeffery G.; Badellino, Karen O; Marchadier, Dawn H; Kempner, Ellis S; Billheimer, Jeffrey; Glick, Jane M; Rader, Daniel JEndothelial lipase (EL) is a member of a subfamily of lipases that act on triglycerides and phospholipids in plasma lipoproteins, which also includes lipoprotein lipase and hepatic lipase. EL has a tropism for high density lipoprotein, and its level of phospholipase activity is similar to its level of triglyceride lipase activity. Inhibition or loss-of-function of EL in mice results in an increase in high density lipoprotein cholesterol, making it a potential therapeutic target. Although hepatic lipase and lipoprotein lipase have been shown to function as homodimers, the active form of EL is not known. In these studies, the size and conformation of the active form of EL were determined. Immunoprecipitation experiments suggested oligomerization. Ultracentrifugation experiments showed that the active form of EL had a molecular weight higher than the molecular weight of a simple monomer but less than a dimer. A construct encoding a covalent head-to-tail homodimer of EL (EL-EL) was expressed and had similar lipolytic activity to EL. The functional molecular weights determined by radiation inactivation were similar for EL and the covalent homodimer EL-EL. We previously showed that EL could be cleaved by proprotein convertases, such as PC5, resulting in loss of activity. In cells overexpressing PC5, the covalent homodimeric EL-EL appeared to be more stable, with reduced cleavage and conserved lipolytic activity. A comparative model obtained using other lipase structures suggests a structure for the head-to-tail EL homodimer that is consistent with the experimental findings. These data confirm the hypothesis that EL is active as a homodimer in head-to-tail conformation.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, RenyuBackground 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.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 A Focused Antibody Library for Selecting scFvs Expressed at High Levels in the Cytoplasm(2007-11-22) Philbert, Pascal; Stoessel, Audrey; Wang, Wei; Sibler, Annie-Paule; Bec, Nicole; Larroque, Christian; Saven, Jeffery G.; Courtête, Jérôme; Weiss, Etienne; Martineau, PierreBackground Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus. Results We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the αβ tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus. Conclusion This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.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 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 Progress in the development and application of computational methods for probabilistic protein design(2004-07-06) Park, Sheldon; Wang, Wei; Boder, Eric T.; Saven, Jeffery G.; Kono, HidetoshiProteins exhibit a wide range of physical and chemical properties, including highly selective molecular recognition and catalysis, and are also key components in biological metabolic, catabolic, and signaling pathways. Given that proteins are well-structured and can now be rapidly synthesized, they are excellent targets for engineering of both molecular structure and biological function. Computational analysis of the protein design problem allows scientists to explore sequence space and systematically discover novel protein molecules. Nonetheless, the complexity of proteins, the subtlety of the determinants of folding, and the exponentially large number of possible sequences impede the search for peptide sequences compatible with a desired structure and function. Directed search algorithms, which identify directly a small number of sequences, have achieved some success in identifying sequences with desired structures and functions. Alternatively, one can adopt a probabilistic approach. Instead of a finite number of sequences, such calculations result in a probabilistic description of the sequence ensemble. In particular, by casting the formalism in the language of statistical mechanics, the site-specific amino acid probabilities of sequences compatible with a target structure may be readily identified. The computational probabilities are well suited for both de novo protein design of particular sequences as well as combinatorial, library-based protein engineering. The computed site-specific amino acid profile may be converted to a nucleotide base distribution to allow assembly of a partially randomized gene library. The ability to synthesize readily such degenerate oligonucleotide sequences according to the prescribed distribution is key to constructing a biased peptide library genuinely reflective of the computational design. Herein we illustrate how a standard DNA synthesizer can be used with only a slight modification to the synthesis protocol to generate a pool of degenerate DNA sequences, which encodes a predetermined amino acid distribution with high fidelity.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 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.
- «
- 1 (current)
- 2
- 3
- »