DOCSLIB.ORG

Adaptation of E. Coli Towards Tryptophan Analog Usage

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Adaptation of E. Coli Towards Tryptophan Analog Usage Adaptation of E. coli towards Tryptophan analog usage vorgelegt von Master of Science in Chemie Stefan Oehm aus Neumarkt i.d.OPf. von der Fakultät II - Mathematik und Naturwissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Naturwissenschaften Dr. rer. nat. genehmigte Dissertation Promotionsausschuss Vorsitzende: Prof. Dr. rer. nat. Maria Andrea Mroginski Gutachter: Prof. Dr. rer. nat. Nediljko Budisa Gutachter: Prof. Dr. rer. nat. Rupert Mutzel Tag der wissenschaftlichen Aussprache: 11.07.2016 Berlin 2016 Look what I have found A seashell in a sea of shells I’m good at planting my own seeds To sprout endless hell It’s dark like Poe Dredg - Planting Seeds Parts of this work were published as listed below Oehm, S., Hösl, M., Peil, L. , Semmler, T., Rappsilber, J., Budisa, N. Mechanisms of an adap- tive evolution experiment towards non-canonical amino acid usage manuscript in preparation Hösl, M. G., Oehm, S., Durkin, P., Peil, L., Rappsilber, J., Rinehart, J., Darmon, E., Leach, D., Söll, D., Budisa, N., Chemical Evolution of a Bacterium’s Proteome Angewandte Chemie - International Edition, 2015, 54(34), 10030-34 Parts of this work were presented as listed below Poster presentation at the Protein Synthesis Meeting, St. Augustine, USA – June 2015 Project talk at the GRK Fluorine graduate school, Berlin, Deutschland – May 2015 Project talk at the COST CM1004 Spring Meeting, Berlin, Deutschland – April 2013 Contents Summary I Zusammenfassung III Abbreviations V List of Figures IX List of Tables XI 1 Introduction 1 1.1 The Genetic Code . 1 1.2 Genetic code engineering . 5 1.2.1 Ambiguous decoding . 5 1.2.2 Stop and sense codon suppression . 7 1.2.3 Codon reassignment . 10 1.2.4 Genetic code reduction . 11 1.3 Xenobiology . 13 1.3.1 Biocontainment through genetically recoded organisms . 14 1.3.2 Chemical evolution of a bacterial genome . 15 1.3.3 Chemical evolution of a bacterial proteome . 16 1.4 Adaptive evolution experiments . 18 1.4.1 General stress response as an target in evolution experiments . 20 2 Aim of the study 23 3 Results 25 3.1 Choice of the analog and activation by its aaRS . 25 3.1.1 Trp and its surrogates . 25 3.1.2 Activation of [3,2]Tpa by the endogenous TrpRS . 27 3.2 Experimental configuration of the starting strains and the growth medium 28 3.2.1 Genetic configurations . 28 3.2.1.1 MT0 . 28 3.2.1.2 TUB00 . 29 3.2.1.3 Differences in strain setup of MT0 and TUB00 . 29 3.2.2 Growth medium . 30 3.3 Adaptive evolution experiment with MT0 . 31 3.3.1 Evaluation of growth in different media of intermediate isolates and the final evolvate MT20 . 32 3.3.2 Analysis of the proteome wide incorporation of β-(thieno[3,2-b]pyrrolyl)- L-alanine ([3,2]Tpa) . 34 3.3.3 Initial characterization of derivates . 36 3.4 Adaptive evolution experiment with TUB00 . 39 3.4.1 Growth behavior of TUB00 and adapted isolates . 41 3.4.2 Proteomic analysis of ancestral and evolved derivatives . 42 3.4.2.1 Knockout of lysA and argA in TUB0 for SILAC analysis . 42 3.4.2.2 Overview of the SILAC experiments . 43 3.4.2.3 Effect of β-thieno[3,2-b]-pyrrole ([3,2]Tp) addition on TUB00 . 45 3.4.2.4 Up- and Downregulation of proteins in the evolved derivatives . 49 3.4.3 Next generation sequencing of TUB13 and its precursors . 53 3.4.3.1 Mutation rate in the AEE . 53 3.4.3.2 Detected mutations in the evolvates . 54 3.4.3.3 Appearance and fixation of selected mutations in the AEE experiment . 57 3.4.4 Analysis of mutations of adapted TUB00 . 59 3.4.4.1 Clustering of mutations . 59 3.4.4.2 Stringent response related mutations . 60 3.4.4.3 Mutations in proteases . 63 3.4.5 Adaptation model derived from Proteomics and Genomics . 64 3.5 Towards Biocontainment of evolved derivatives . 67 3.5.1 Genetic replacement system . 67 3.5.2 PylRS-library construction . 68 3.5.3 Establishment of a genetic replacement system for tryptophan and analogs 71 4 Conclusion and Outlook 75 5 Materials 77 5.1 Media and supplements . 77 5.2 Microoganisms . 80 5.3 Buffers and solutions . 81 5.3.1 Primer and Plasmids . 83 5.3.1.1 Primer . 83 5.3.1.2 Plasmids . 85 6 Methods 87 6.1 Molecular biological methods . 87 6.2 Microbiological methods . 90 6.3 Protein expression and purification . 93 6.4 Enzymatic synthesis of [3,2]Tpa . 95 6.5 Biochemical methods . 95 6.6 Analytical methods . 96 6.7 Bioinformatic analysis . 100 7 Appendix 101 7.1 Adaptive evolution experiment with MT0 and TUB00 . 101 7.1.1 Defining metabolic blocks . 101 7.1.2 Medium composition MT0-experiment . 102 7.1.3 Medium composition TUB00-experiment . 103 7.1.4 Mutations found in MT18 by NGS . 104 7.2 Amino acid analysis via GC-MS . 105 7.2.1 GC-MS spectra of derivatized Trp . 105 7.2.2 Chromatogram of hydrolyzed and derivatized E. coli proteome . 106 7.3 Proteomics . 108 7.3.1 Regulators . 109 7.3.1.1 Effect of [3,2]Tp in TUB00 . 109 7.3.1.2 Evolvates TUB10 and TUB13 . 116 7.4 SDS-PAGE of expressed proteins . 123 Bibliography 125 Summary The presented study set out to change the amino acid set of the genetic code in two different Trp-auxotrophic E. coli strains with the help of the serial transfer regime under constant evolutionary pressure. By increasing the stringency of the system as the source for the canonical amino acid is depleted, the bacteria were forced to incorporate the Trp- surrogate [3,2]Tpa at all Trp-positions in the proteome. Both evolvates of the ancestral strains MT0 and TUB00 are capable of surviving the Trp → [3,2]Tpa replacement, but still show a preference for their canonical substrate. At regular time points, a representative sample of the population was frozen and made available for later analysis of the trajectory of the evolution. MS/MS and amino acid analysis via GC-MS were conducted and proved the successful exchange of all Trp by [3,2]Tpa. While the strain configuration of MT0 hampered a comprehensive analysis of the evolu- tionary trajectory, recent advances in genome sequencing (next generation sequencing) delivered information about the genetic changes of the evolved derivatives of the TUB cell line. To get a complete picture of the cellular response to the exchange of Trp, proteomic data were collected and analyzed. It turned out that the addition of the translationally active Trp-analog [3,2]Tpa caused a state of elevated stress response, most likely due to the production of a destabilized proteome. This hampers the growth of the ancestral strain in the presence of [3,2]Tpa. During the cultivation in the serial transfer regime, the strain adapts in a way to rearrange its expression profile to an unstressed ground state. The proteomic results are validated and allegeable by a key mutation in σS found by whole genome sequencing. This study presents the first analytically unambiguous exchange of a canonical amino acid by a synthetic analog throughout the whole proteome. All UGG-positions are Trp decoded by [3,2]Tpa-tRNACCA and result in a functional proteome. Additionally, the study presents an adaptation mechanism based on a shutdown stress response system and shows insights into proteome-wide replacements of canonical amino acids. I Zusammenfassung Die vorliegende Arbeit befasste sich mit der Veränderung des Aminosäurerepertoires des genetischen Codes innerhalb zwei verschiedener Trp-auxotropher E. coli Stämme mit Hilfe einer seriellen Übertragungsstrategie unter ständigem evolutionären Druck. Durch Erhöhung der Stringenz des Systems durch Entziehung der kanonischen Aminosäure wurden die Stämme gezwungen, das Trp-Analog [3,2]Tpa an allen Trp-Positionen im Proteom einzubauen. Beide Evolvate der Ausgangstämme MT0 und TUB00 können den Trp → [3,2]Tpa Austausch überleben, zeigen jedoch immer noch Präferenz für das kanonische Substrat. In regelmäßigen Abständen des Experiments, wurde ein repräsenta- tiver Teil der Population für eine spätere Analyse des evolutionären Verlaufs aufbewahrt. MS/MS und GC-MS Experimente wurden durchgeführt, um die erfolgreiche Substitu- tion aller Trp durch [3,2]Tpa zu beweisen. Während die Stammkonfiguration des MT0 eine nähere Analyse des Verlaufs der Adaptation behinderte, offenbarten Genomse- quenzierungen von Isolaten aus der TUB Population die genetischen Veränderungen, die für die Anpassung an das synthetische Medium verantwortlich sind. Um ein voll- ständiges Bild der zellulären Antwort auf den Austausch von Trp durch die synthetische Aminosäure zu erhalten, wurde das Expressionsprofil des Ausgangstammes und der Evolvate mittels SILAC untersucht. Es stellte sich heraus, dass das translational-aktive Trp-Analog [3,2]Tpa eine erhöhte Stressantwort, vermutlich aufgrund eines destabil- isierten Proteoms, hervorruft. Dies verhindert das Wachstum des Mutterstammes in Anwesenheit von [3,2]Tpa. Im Verlauf des Langzeitkultivierungs-Experiments, adaptiert sich der Stamm, indem er sein Expressionsprofil auch in Anwesenheit von [3,2]Tpa auf den relaxierten Grundzustand anpasst. Diese Proteomdaten können von einer mittels Genomsequenzierung gefunden Mutation in σS erklärt werden. Diese Studie zeigt den ersten analytisch fundierten, proteomweiten Austausch einer kanonischen Aminosäure durch ein synthetisches Analog. Alle UGG-Positionen werden Trp mit einer [3,2]Tpa-tRNACCA gelesen. Des Weiteren wurde ein Adaptationsmechanismus gefunden, der auf einem Ausschalten der Stressantwort des Bakteriums beruht. Hiermit wurden neue Einblicke in den Proteom-weiten Austausch von Aminosäuren erhalten. III Abbreviations A adenine Amp ampicillin ATP adenosine triphosphate aa amino acid aaRS amino acyl tRNA-synthetase cgOAHSS O-acetylhomoserine sulfhydrylase 2aza-Trp 2-aza-L-tryptophan 7aza-Trp 7-aza-L-tryptophan Aha azidohomoalanine pAzF p-azido-L-phenylalanine B.
Load more

Recommended publications
  • Backbone Dynamics of Free Barnase and Its Complex with Barstar Determined by 15N NMR Relaxation Study
    Journal of Biomolecular NMR, 18: 107–118, 2000. KLUWER/ESCOM 107 © 2000 Kluwer Academic Publishers. Printed in the Netherlands. Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study Sarata C. Sahua, Abani K. Bhuyanb, Jayant B. Udgaonkarb & R.V. Hosura;∗ aDepartment of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005, India; bNational Centre for Biological Sciences, Tata Institute of Fundamental Research, University of Agricultural Sciences (UAS) - Gandhi Krishi Vigyan Kendra (GKVK) Campus, Bangalore 560 065, India Received 18 April 2000; Accepted 7 July 2000 Key words: backbone dynamics, barnase, barstar, complex, 15N NMR relaxation, protein–protein interactions Abstract Backbone dynamics of uniformly 15N-labeled free barnase and its complex with unlabelled barstar have been studied at 40 ◦C, pH 6.6, using 15N relaxation data obtained from proton-detected 2D {1H}-15N NMR spec- 15 troscopy. N spin-lattice relaxation rate constants (R1), spin-spin relaxation rate constants (R2), and steady-state heteronuclear {1H}-15N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N- terminal residue) backbone amide 15N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (τm) are 5.0 and 9.5 ns for the free and complexed barnase, respectively.
    [Show full text]
  • Characterization of in Vitro Oxidized Barstar
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector FEBS 15911 FEBS Letters 370 (1995) 273-277 Characterization of in vitro oxidized barstar C. Frisch, G. Schreiber, A.R. Fersht* Cambridge Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge, CB2 2QH, UK Received 13 July 1995 mutant which has been solved independently by Guillet et al. Abstract The polypeptide inhibitor of the ribonuclease barnase, [8] to 2.6 A resolution and by Buckle et al. [4] to 2.0/k resolu- barstar, has two cysteine residues in positions 40 and 82. These tion. The distance between the s-carbon atoms of the alanines have been proposed to form a disulfide bridge leading to an in position 40 and 82 in the two crystal structures of the barstar increase in stability without changing the inhibitory activity of the protein. Barstar and a mutant (E80A) were oxidized in vitro and (C40A/C82A) double mutant is 11.4 A [8] and 11.7 A [4], re- the biochemical and physico-chemical properties of the oxidized spectively. In the solution structure of barstar that was deter- monomers were analysed. The oxidized proteins show no inhibi- mined by NMR [7] (Fig. 1), the distance between the s-carbon tion of barnase using a plate assay and are significantly destabi- atoms of the cysteines is 11.5 h. These s-carbon distances are lized. CD spectra indicate a loss of secondary structure. The outside the normal range for a disulfide bond, which is usually amino acid substitution E80 --~ A stabilizes the oxidized barstar from 4.4 to 6.8 A [9].
    [Show full text]
  • The Folding of Groel-Bound Barnase As a Model for Chaperonin-Mediated Protein Folding (Chaperone/Protein Engineering)
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5326-5330, June 1995 Biochemistry The folding of GroEL-bound barnase as a model for chaperonin-mediated protein folding (chaperone/protein engineering) FERNANDO J. CORRALES AND ALAN R. FERSHT* Medical Research Council Unit for Protein Function and Design and Cambridge Centre for Protein Engineering, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 lEW, United Kingdom Contributed by Alan R. Fersht, March 3, 1995 ABSTRACT We have analyzed the pathway of folding of solution, then the rate of folding should fall to zero when all barnase bound to GroEL to resolve the controversy ofwhether the barnase becomes bound at saturating concentrations of proteins can fold while bound to chaperonins (GroEL or GroEL. Cpn6O) or fold only after their release into solution. Four We now analyze the folding pathway in detail by stopped- phases in the folding were detected by rapid-reaction kinetic flow monitoring the change of tryptophan fluorescence of measurements of the intrinsic fluorescence of both wild type barnase, GroEL not having tryptophan residues. Unfortu- and barnase mutants. The phases were assigned from their nately, the fluorescence of denatured barnase when bound to rate laws, sensitivity to mutations, and correspondence to GroEL is so close to that of fully folded barnase that it is regain ofcatalytic activity. At high ratios ofdenatured barnase difficult to monitor the folding by using conventional stopped- to GroEL, 4 mol ofbarnase rapidly bind per 14-mer of GroEL. flow fluorimeters. We have now overcome these difficulties by At high ratios of GroEL to barnase, 1 mol of barnase binds replacing Trp-94 by tyrosine (mutant W94Y), which removes with a rate constant of 3.5 x 107 s-1 M-1.
    [Show full text]
  • Investigating the Structural Determinants of Electrostatic Binding Among Protein-Protein Complexes: a Systematic, Large-Scale Computational Study
    Investigating the Structural Determinants of Electrostatic Binding among Protein-protein Complexes: A Systematic, Large-scale Computational Study Emma Nechamkin April 2012 Wellesley College Wellesley, MA Submitted in Partial Fulfillment of the Prerequisites for Honors in Chemistry Acknowledgements Writing my senior thesis has been a wonderful experience, and there are many people to whom I owe thanks for support and guidance. First and foremost, my brilliant advisor Mala Radhakrishnan has been incredibly supportive and understanding. In dorkier terms, her advising style is incredibly close to the hypothetical optimum. Without her constant guidance, motivation, and feed- back, my thesis simply would not exist. I am truly grateful to be a student in her lab. Second, the faculty on my thesis committee, David Haines and Don Elmore of the Chemistry Department, and YuJin Ko of the English Department, have dedicated significant time to my thesis. In particular, the chemistry faculty on my committee have provided insight on much of my work and offered much needed encouragement. Third, I would like to thank the entire Wellesley College Chemistry Department. Clearly the best department on campus, the chemistry department has an unparal- leled involvement with and dedication to its students. At a sometimes stressful and overwhelming school like Wellesley, I am glad to have found my niche. Fourth, each member of the Radhakrishnan Lab over my time here has taught me something about research and listened to at least one incredibly long story I have shared. For the group's helpfulness, reassurance, and companionship, I am deeply grateful. In particular, Ying Yi Zhang has shared some wonderful code with me.
    [Show full text]
  • FOR Environmental Release of Genetically Engineered Mustard
    Assessment of Food and Environmental Safety of GE mustard ASSESSMENT OF FOOD AND ENVIRONMENTAL SAFETY (AFES) FOR Environmental release of Genetically Engineered Mustard (Brassica juncea) hybrid DMH-11 and use of parental events (Varuna bn3.6 and EH2 modbs2.99) for development of new generation hybrids Application submitted by Centre for Genetic Manipulation of Crop Plants (CGMCP), University of Delhi South Campus, New Delhi 2016 1 Assessment of Food and Environmental Safety of GE mustard 2 Assessment of Food and Environmental Safety of GE mustard Table of Contents 1 CHAPTER 1: INTRODUCTION 5 1.1 The Application 6 1.2 Global status of hybrid seed production technology in Brassica napus using MS-RF system deploying the genes used for B. juncea 8 2 CHAPTER 2: BIOLOGY OF INDIAN MUSTARD 11 2.1 Origin and domestication of Brassica juncea 11 2.2 Brassica species present in India and their distribution 12 2.3 Cultivation of B. juncea: soil and climate requirement 14 2.4 Standard agriculture practices for growing B. juncea 14 2.5 Weeds, Major pest and disease 15 2.6 Zonalization of varietal testing 15 3 CHAPTER 3: INDIAN BIOSAFETY REGULATORY FRAMEWORK 17 3.1 Introduction 17 3.2 Step by step process to be followed by the applicant 22 3.3 Step by step regulatory compliance and data generation in the case of GE mustard parental lines and hybrid DMH-11 24 3.4 Assessment of Food/feed and Environmental Safety (AFES) - Risk Assessment Process 25 4 CHAPTER 4: MOLECULAR CHARACTERIZATION OF GE MUSTARD HYBRID DMH-11 AND ITS PARENTAL LINES 33 4.1 Introduction 33 4.2 The male sterility- fertility restorer technology 34 4.3 Gene constructs 35 4.4 Method of genetic transformation of B.
    [Show full text]
  • A General Approach for the Generation of Orthogonal Trnas
    Chemistry & Biology 8 (2001) 883^890 www.elsevier.com/locate/chembiol Research Paper A general approach for the generation of orthogonal tRNAs Lei Wang a; 1, Peter G. Schultz b; * aDepartment of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA bDepartment of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Received 26 March 2001; accepted 29 May 2001 First published online 27 July 2001 Abstract Background: The addition of new amino acids to the genetic cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four code of Escherichia coli requires an orthogonal suppressor tRNA mutant suppressor tRNAs were selected that are poorer substrates Tyr that is uniquely acylated with a desired unnatural amino acid by for E. coli synthetases than M. jannaschii tRNACUA, but still can Tyr an orthogonal aminoacyl-tRNA synthetase. A tRNACUA^tyrosyl- be charged efficiently by M. jannaschii TyrRS. Tyr tRNA synthetase pair imported from Methanococcus jannaschii Conclusions: The mutant suppressor tRNACUA together with can be used to generate such a pair. In vivo selections have been the M. jannaschii TyrRS is an excellent orthogonal tRNA^ developed for selecting mutant suppressor tRNAs with enhanced synthetase pair for the in vivo incorporation of unnatural amino orthogonality, which can be used to site-specifically incorporate acids into proteins. This general approach may be expanded to unnatural amino acids into proteins in E. coli. generate additional orthogonal tRNA^synthetase pairs as well as Results: A library of amber suppressor tRNAs derived from M. probe the interactions between tRNAs and their cognate Tyr Tyr jannaschii tRNA was generated.
    [Show full text]
  • Part II – Summary
    Bayer CropScience AG EFSA-GMO-BE-2010-81 Page 1 of 22 15 September 2011 Part II – Summary PART II Request for Authorization of Glufosinate Ammonium-Tolerant Genetically Modified Oilseed Rape MS8, RF3 and MS8xRF3 in accordance with article 5 of Regulation (EC) N° 1829/2003 A. GENERAL INFORMATION 1. Details of application a) Member State of application: Belgium b) Application number: EFSA-GMO-BE-2010-81 c) Name of the product (commercial and other names): Glufosinate ammonium-tolerant genetically modified MS8, RF3 and MS8xRF3 oilseed rape. The unique identifiers assigned to MS8/RF31 oilseed rape are: MS8: ACS-BNØØ5-8 RF3: ACS-BNØØ3-6 MS8xRF3: ACS-BNØØ5-8xACS-BNØØ3-6 d) Date of acknowledgement of valid application: Not available at the date of application 2. Applicant a) Name of applicant: This is an application submitted by Bayer CropScience AG. b) Address of applicant: Bayer CropScience AG represented by Bayer BioScience NV Alfred-Nobel-Strasse 50 Technologiepark 38 D - 40789 Monheim am Rhein B-9052 Gent Germany Belgium c) Name and address of the person established in the Community who is responsible for the placing on the market, whether it be the manufacturer, the importer or the distributor, if different from the applicant (Commission Decision 2004/204/EC Art 3(a)(ii)): MS8/RF3 oilseed rape will be imported and processed in the EU by the same groups who currently import, 1 The designation “MS8/RF3” is used to refer to “MS8, RF3 and MS8xRF3”. Bayer CropScience AG EFSA-GMO-BE-2010-81 Page 2 of 22 15 September 2011 Part II – Summary process and distribute commodity oilseed rape.
    [Show full text]
  • Identification and Characterization of Protein Folding Intermediates Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini-Allocatelli
    Identification and characterization of protein folding intermediates Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini-Allocatelli To cite this version: Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini-Allocatelli. Identifica- tion and characterization of protein folding intermediates. Biophysical Chemistry, Elsevier, 2007, 128 (2-3), pp.105. 10.1016/j.bpc.2007.04.008. hal-00501662 HAL Id: hal-00501662 https://hal.archives-ouvertes.fr/hal-00501662 Submitted on 12 Jul 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ÔØ ÅÒÙ×Ö ÔØ Identification and characterization of protein folding intermediates Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini- Allocatelli PII: S0301-4622(07)00087-7 DOI: doi: 10.1016/j.bpc.2007.04.008 Reference: BIOCHE 4952 To appear in: Biophysical Chemistry Received date: 29 March 2007 Revised date: 16 April 2007 Accepted date: 16 April 2007 Please cite this article as: Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini-Allocatelli, Identification and characterization of protein folding inter- mediates, Biophysical Chemistry (2007), doi: 10.1016/j.bpc.2007.04.008 This is a PDF file of an unedited manuscript that has been accepted for publication.
    [Show full text]
  • Spectrin SH3 Domain Xavier Periole,1* Michele Vendruscolo,2 and Alan E
    proteins STRUCTURE O FUNCTION O BIOINFORMATICS Molecular dynamics simulations from putative transition states of a-spectrin SH3 domain Xavier Periole,1* Michele Vendruscolo,2 and Alan E. Mark1,3,4* 1 Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9747 AG Groningen, The Netherlands 2 Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom 3 School of Molecular and Microbial Sciences, University of Queensland, St Lucia, 4072, Queensland, Australia 4 Institute for Molecular Biosciences, University of Queensland, St Lucia, 4072, Queensland, Australia INTRODUCTION ABSTRACT Understanding of the process of protein folding is one of the grand challenges in A series of molecular dynam- molecular biology. Ever since it was shown that the information contained in the ics simulations in explicit sol- sequence of amino acids is sufficient for a protein to find the structure of its native vent were started from nine state,1 experimentalists and theoreticians have tried to understand the mechanisms of structural models of the tran- this important biological process.2–5 sition state of the SH3 do- 5 main of a-spectrin, which Much effort has been focused on proteins that undergo two-state folding. The pri- were generated by Lindorff- mary advantage of two-state proteins is the lack of detectable intermediate states, so Larsen et al. (Nat Struct Mol their folding process can be considered as involving a transition from a broad ensem- Biol 2004;11:443–449) using ble of configurations representing the unfolded state to a narrow ensemble of configu- molecular dynamics simula- rations making up the native state, via a specific transition state.
    [Show full text]
  • Recombinant Expression of Barnase in Escherichia Coli and Its
    Shankar et al. Microb Cell Fact (2021) 20:171 https://doi.org/10.1186/s12934-021-01642-y Microbial Cell Factories RESEARCH Open Access Recombinant expression of Barnase in Escherichia coli and its application in plasmid purifcation Ram Shankar1* , Nina Schäfer1, Marco Schmeer1, Joe Max Risse2, Karl Friehs2 and Martin Schleef1,2,3* Abstract Background: The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purif- cation. As the feld of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purifcation protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. Results: The pure enzyme without the inhibitor barstar was exported to the extracellular space through the peri- plasm and then purifed from the cell-free supernatant. Cation exchange chromatography was employed as a primary purifcation step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units 1 mL− ), in principle its application to plasmid DNA purifcation could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. Conclusions: The current work focusses on the downstream purifcation strategies for a recombinant RNase and sets a framework for higher scale production if specifc productivity is increased by optimal hosts and/or re-engineered plasmids.
    [Show full text]
  • Molecular Constructor on the Basis of Barnase–Barstar Module
    ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 6, pp. 685–701. © Pleiades Publishing, Ltd., 2009. Original Russian Text © S.M. Deev, E.N. Lebedenko, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 6, pp. 761–778. Antibody Engineering: Molecular Constructor on the basis of Barnase–Barstar Module S. M. Deev1 and E. N. Lebedenko Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia Received May 19, 2009; in final form, May 29, 2009 Abstract—Today, antibody engineering for clinical applications is a rapidly progressing field of science and a big business. The basic functions of an antibody can be spatially differentiated and attributed to various struc- tural domains of a molecule. Therefore, each of them may be an object for engineering with the aim of using a definite antibody function. In this sense, the potential of antibodies is unique. In this article, recent achieve- ments and current problems of antibody engineering are briefly reviewed. The main attention is focused on a molecular constructor that allows for obtaining, with the help of a versatile barnase–barstar module, mono- and multivalent miniantibodies and their derivatives with outlined properties. Key words: single chain antibodies, multivalency, bispecificity, targeted delivery, barnase–barstar module DOI: 10.1134/S1068162009060041 INTRODUCTION mAbs), active or diagnostic agents, for instance, cytok- ines, protein toxins and radioisotopes, enzymes, fluo- Currently, antibodies have the second largest pro- rescent proteins, etc. duction value in the pharmaceutical market after vac- cines. More than 85% of antibodies permitted for clin- The current review will focus on a molecular con- ical use are products of antibody engineering.
    [Show full text]
  • Using Stochastic Roadmap Simulation to Predict Experimental Quantities in Protein Folding Kinetics: Folding Rates and Phi-Values
    Using Stochastic Roadmap Simulation to Predict Experimental Quantities in Protein Folding Kinetics: Folding Rates and Phi-Values Tsung-Han Chiang∗ Mehmet Serkan Apaydin† Douglas L. Brutlag‡ David Hsu∗ Jean-Claude Latombe‡ ∗National University of Singapore, Singapore, 117543, Singapore †Duke University, Durham, NC, 27705, USA ‡Stanford University, Stanford, CA 94305, USA Abstract This paper presents a new method for studying protein folding kinetics. It uses the recently intro- duced Stochastic Roadmap Simulation (SRS) method to estimate the transition state ensemble (TSE) and predict the rates and the Φ-values for protein folding. The new method was tested on 16 proteins, whose rates and Φ-values have been determined experimentally. Comparison with experimental data shows that our method estimates the TSE much more accurately than an existing method based on dynamic pro- gramming. This improvement leads to better folding-rate predictions. We also compute the mean first passage time of the unfolded states and show that the computed values correlate with experimentally de- termined folding rates. The results on Φ-value predictions are mixed, possibly due to the simple energy model used in the tests. This is the first time that results obtained from SRS have been compared against a substantial amount of experimental data. The results further validate the SRS method and indicate its potential as a general tool for studying protein folding kinetics. 1 Introduction Protein folding is a fundamental biological process. Starting out as a long, linear chain of amino acids, a protein molecule remarkably configures itself, or folds, into a unique three-dimensional structure, called the native conformation, in order to perform vital biological functions.
    [Show full text]