Brad L Pentelute

Harvard University, Cambridge, Massachusetts, United States

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Publications (26)160.1 Total impact

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    ABSTRACT: We have visualized by cryo-electron microscopy (cryo-EM) the complex of the anthrax protective antigen (PA) translocon and the N-terminal domain of anthrax lethal factor (LFN ) inserted into a nanodisc model lipid bilayer. We have determined the structure of this complex at a nominal resolution of 16Å by single-particle analysis and three-dimensional reconstruction. Consistent with our previous analysis of negatively stained unliganded PA, the translocon comprises a globular structure (cap) separated from the nanodisc bilayer by a narrow stalk that terminates in a transmembrane channel (incompletely distinguished in this reconstruction). The globular cap is larger than the unliganded PA pore, probably due to distortions introduced in the previous negatively stained structures. The cap exhibits larger, more distinct radial protrusions, previously identified with PA domain three, fitted by elements of the NMFF PA prepore crystal structure. The presence of LFN , though not distinguished due to the seven-fold averaging used in the reconstruction, contributes to the distinct protrusions on the cap rim volume distal to the membrane. Furthermore, the lumen of the cap region is less resolved than the unliganded negatively stained PA, due to the low contrast obtained in our images of this specimen. Presence of the LFN extended helix and N terminal unstructured regions may also contribute to this additional internal density within the interior of the cap. Initial NMFF fitting of the cryoEM-defined PA pore cap region positions the Phe clamp region of the PA pore translocon directly above an internal vestibule, consistent with its role in toxin translocation.
    Full-text · Article · May 2013 · Protein Science
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    ABSTRACT: We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LF(N) ) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (∼43%). Three-dimensional analysis of negatively stained single particles revealed the LF(N) -PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LF(N) molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LF(N) -PA nanodisc structures and use of advanced automated particle selection methods.
    Full-text · Article · Apr 2013 · Protein Science
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    ABSTRACT: 'Chemical ligation'--the regioselective and chemoselective covalent condensation of unprotected peptide segments--has enabled the synthesis of polypeptide chains of more than 200 amino acids. An efficient total chemical synthesis of the insulin molecule has been devised on the basis of a key ester-linked intermediate that is chemically converted to fully active human insulin. Enzyme molecules of defined covalent structure and with full enzymatic activity have been prepared and characterized by high-resolution X-ray crystallography. A 'glycoprotein mimetic' of defined chemical structure and with a mass of 50,825 Da, has been prepared and shown to have full biological activity and improved pharmacokinetic properties. D-protein molecules that are the mirror images of proteins found in the natural world have been prepared by total chemical synthesis. Racemic protein mixtures, consisting of the D-enantiomers and L-enantiomers of a protein molecule, form highly ordered centrosymmetric crystals with great ease; this has enabled the determination of the crystal structures of recalcitrant protein molecules. A protein with a novel linear-loop covalent topology of the peptide chain has been designed and synthesized and its structure determined by facile crystallization as the quasi-racemate with the D-form of the native protein molecule.We have developed an optimized total chemical synthesis of biologically active vascular endothelial growth factor-A; total synthesis of the mirror-image protein will be used to systematically develop D-protein antagonists of this important growth factor. The total chemical synthesis of proteins is now a practical reality and enables access to a new world of protein molecules.
    Full-text · Article · Sep 2012 · Journal of Peptide Science
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    ABSTRACT: Lassoed at the other end: Original synthetic and structure determination methods were used to make a protein molecule with an unprecedented linear-loop polypeptide chain topology, and to characterize its X-ray structure.
    Full-text · Article · Feb 2012 · Angewandte Chemie International Edition
  • Suhuai Liu · Brad L. Pentelute · Stephen B. H. Kent
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    ABSTRACT: Ein neuer Weg zu EPO: Mit einer konvergenten chemischen Ligationsstrategie wurde nichtglycosyliertes menschliches Erythropoetin (EPO) hergestellt. Das synthetische [Lysin24, 38, 83]-EPO-Analogon, das über HPLC gereinigt wurde und das korrekte Molekulargewicht hat (siehe Bild), hat eine wohldefinierte kovalente Struktur, ist korrekt gefaltet und zeigt biologische Aktivität in einem Assay mit einer faktorabhängigen Zell-Linie.
    No preview · Article · Jan 2012 · Angewandte Chemie
  • Michael R Sawaya · Brad L Pentelute · Stephen B H Kent · Todd O Yeates
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    ABSTRACT: Racemic protein crystallography offers two key features: an increased probability of crystallization and the potential advantage of phasing centric diffraction data. In this study, a phasing strategy is developed for the scenario in which a crystal is grown from a mixture in which anomalous scattering atoms have been incorporated into only one enantiomeric form of the protein molecule in an otherwise racemic mixture. The structure of a protein crystallized in such a quasi-racemic form has been determined in previous work [Pentelute et al. (2008), J. Am. Chem. Soc. 130, 9695-9701] using the multiwavelength anomalous dispersion (MAD) method. Here, it is shown that although the phases from such a crystal are not strictly centric, their approximate centricity provides a powerful way to break the phase ambiguity that ordinarily arises when using the single-wavelength anomalous dispersion (SAD) method. It is shown that good phases and electron-density maps can be obtained from a quasi-racemic protein crystal based on single-wavelength data. A prerequisite problem of how to establish the origin of the anomalous scattering substructure relative to the center of pseudo-inversion is also addressed.
    No preview · Article · Jan 2012 · Acta Crystallographica Section D Biological Crystallography
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    Suhuai Liu · Brad L Pentelute · Stephen B H Kent
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    ABSTRACT: A new route to EPO: Nonglycosylated human erythropoietin (EPO) was prepared using a convergent chemical ligation synthetic strategy. The synthetic [Lysine(24, 38, 83) ] EPO analogue, which was purified by HPLC and has the correct molecular weight, shows well-defined covalent structure, is correctly folded, and is biologically active in a factor-dependent cell line assay.
    Preview · Article · Dec 2011 · Angewandte Chemie International Edition
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    Audrey Fischer · Matthew A Holden · Brad L Pentelute · R John Collier
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    ABSTRACT: Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.
    Full-text · Article · Sep 2011 · Proceedings of the National Academy of Sciences
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    Joongoo Lee · Yoonjin Kwon · Brad L Pentelute · Duhee Bang
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    ABSTRACT: Since the introduction of kinetically controlled ligation (KCL), a chemoselective reaction between a peptide-(α)thioarylester and a Cys-peptide-(α)thioalkylester, KCL has been utilized for the total chemical synthesis of large proteins (i.e., lysozyme and HIV-protease) by providing fully convergent synthetic routes. Although KCL has the potential to become an important chemistry for protein synthesis, the principle of KCL is not fully characterized. In particular, prior work on KCL has focused on the reactivity difference of the two different -(α)thioester forms-alkyl vs aryl. Another equally important feature of KCL, Xaa-Cys ligation sites, has not been investigated. The work reported here describes combinatorial KCL reactions using model peptides to dissect the interplay of the Xaa(1), Xaa(2), -(α)thioarylester, and -(α)thioalkylester. Results from these studies provide fundamental insights into the KCL reaction, and will lead to the optimal synthetic route for the routine chemical synthesis of large target protein molecules.
    Preview · Article · Aug 2011 · Bioconjugate Chemistry
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    Brad L Pentelute · Onkar Sharma · R John Collier
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    ABSTRACT: Durch die Pore: Ersetzt man saure Aminosäurereste in der N-terminalen Domäne des Letalfaktors von Anthraxtoxin durch Cysteinsäure, wird die Proteintranslokation blockiert, während die Einführung von D-Aminosäuren oder einer alternierenden Lys-Glu-Sequenz keinerlei Effekt hat. Das heißt, dass die Translokation weder stereospezifisch noch strikt sequenzabhängig ist, dafür aber vom Ladungszustand saurer Aminosäurereste abhängt.
    Preview · Article · Mar 2011 · Angewandte Chemie International Edition
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    ABSTRACT: Here we report the total synthesis of kaliotoxin by 'one pot' native chemical ligation of three synthetic peptides. A racemic mixture of D- and L-kaliotoxin synthetic protein molecules gave crystals in the centrosymmetric space group P1 that diffracted to atomic-resolution (0.95 Å), enabling the X-ray structure of kaliotoxin to be determined by direct methods.
    No preview · Article · Sep 2010 · Chemical Communications
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    ABSTRACT: Many bacterial toxins act by covalently altering molecular targets within the cytosol of mammalian cells and therefore must transport their catalytic moieties across a membrane. The Protective-Antigen (PA) moiety of anthrax toxin forms multimeric pores that transport the two enzymatic moieties, the Lethal Factor (LF) and the Edema Factor, across the endosomal membrane to the cytosol. The homologous PA-binding domains of these enzymes contain N-terminal segments of highly charged amino acids that are believed to enter the pore and initiate N- to C-terminal translocation. Here we describe a semisynthesis platform that allows chemical control of this segment in LF(N), the PA-binding domain of LF. Semisynthetic LF(N) was prepared in milligram quantities by native chemical ligation of synthetic LF(N)(14-28)alphathioester with recombinant N29C-LF(N)(29-263) and compared with two variants containing alterations in residues 14-28 of the N-terminal region. The properties of the variants in blocking ion conductance through the PA pore and translocating across planar phospholipid bilayers in response to a pH gradient were consistent with current concepts of the mechanism of polypeptide translocation through the pore. The semisynthesis platform thus makes new analytical approaches available to investigate the interaction of the pore with its substrates.
    Full-text · Article · Feb 2010 · ACS Chemical Biology
  • Stephen Kent · Brad Pentelute · Duhee Bang · Erik Johnson · Thomas Durek
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    ABSTRACT: The present invention concerns methods and compositions for synthesizing a polypeptide using kinetically controlled reactions involving fragments of the polypeptide for a fully convergent process. In more specific embodiments, a ligation involves reacting a first peptide having a protected cysteyl group at its N-terminal and a phenylthioester at its C-terminal with a second peptide having a cysteine residue at its N-termini and a thioester at its C-termini to form a ligation product. Subsequent reactions may involve deprotecting the cysteyl group of the resulting ligation product and/or converting the thioester into a thiophenylester.
    No preview · Article · Jan 2010
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    ABSTRACT: We describe the use of racemic crystallography to determine the X-ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers L-plectasin and D-plectasin were prepared by total chemical synthesis; interestingly, L-plectasin showed the expected antimicrobial activity, while D-plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X-ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group P1 with one L-plectasin molecule and one D-plectasin molecule forming the unit cell. Dimer-like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%-15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation.
    Full-text · Article · Jun 2009 · Protein Science
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    ABSTRACT: Racemic protein crystallography, enabled by total chemical synthesis, has allowed us to determine the X-ray structure of native scorpion toxin BmBKTx1; direct methods were used for phase determination. This is the first example of a protein racemate that crystallized in space group I41/a.
    Preview · Article · Feb 2009 · Journal of the American Chemical Society
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    ABSTRACT: Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 A. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.
    Full-text · Article · Aug 2008 · Journal of the American Chemical Society
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    ABSTRACT: The recently discovered glycine-rich snow flea antifreeze protein (sfAFP) has no sequence homology with any known proteins. No experimental structure has been reported for this interesting protein molecule. Here we report the total chemical synthesis of the mirror image forms of sfAFP (i.e., L-sfAFP, the native protein, and D-sfAFP, the native protein's enantiomer). The predicted 81 amino acid residue polypeptide chain of sfAFP contains Cys residues at positions 1, 13, 28, and 43 and was prepared from four synthetic peptide segments by sequential native chemical ligation. After purification, the full-length synthetic polypeptide was folded at 4 degrees C to form the sfAFP protein containing two disulfides. Chemically synthesized sfAFP had the expected antifreeze activity in an ice recrystallization inhibition assay. Mirror image D-sfAFP protein was prepared by the same synthetic strategy, using peptide segments made from d-amino acids, and had an identical but opposite-sign CD spectrum. As expected, D-sfAFP displays the same antifreeze properties as L-sfAFP, because ice presents an achiral surface for sfAFP binding. Facile synthetic access to sfAFP will enable determination of its molecular structure and systematic elucidation of the molecular basis of the antifreeze properties of this unique protein.
    Full-text · Article · Aug 2008 · Journal of the American Chemical Society
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    ABSTRACT: Chemistry for biology: IGF-1 and [Gly7D-Ala]IGF-1 have been prepared by a total chemical synthesis and their folding and receptor-binding properties compared (see scheme). This approach facilitates the introduction of nonstandard amino acids whose biosynthetic incorporation is difficult or not feasible and thus promises to lead to a new generation of structure–activity studies of potential interest in the treatment of cancer and diabetes mellitus.
    Preview · Article · Feb 2008 · Angewandte Chemie International Edition
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    ABSTRACT: Chemie für Biologen: IGF-1 und [Gly7D-Ala]IGF-1 wurden durch eine chemische Totalsynthese hergestellt, und ihre Faltung und Rezeptorbindung wurden verglichen (siehe Schema). Dieser Ansatz erleichtert den Einbau ungewöhnlicher Aminosäuren, der auf biosynthetischem Weg problematisch oder unmöglich ist. Daraus ergeben sich Perspektiven für Struktur-Aktivitäts-Studien mit möglichen Auswirkungen auf die Krebs- und Diabetes-mellitus-Therapie.
    No preview · Article · Dec 2007 · Angewandte Chemie
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    ABSTRACT: The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-A resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.
    Full-text · Article · Nov 2007 · Journal of Molecular Biology

Publication Stats

725 Citations
160.10 Total Impact Points

Institutions

  • 2011-2013
    • Harvard University
      Cambridge, Massachusetts, United States
  • 2010-2013
    • Harvard Medical School
      • Department of Microbiology and Immunobiology
      Boston, Massachusetts, United States
  • 2006-2012
    • University of Chicago
      • • Department of Chemistry
      • • Department of Biochemistry & Molecular Biology
      Chicago, Illinois, United States