https://doi.org/10.17192/z2012.0662 opus:4077 2012-01-30 application/pdf Chemistry + allied sciences Chemie ppn:309913268 Proteine 2012-10-05 Chemie Biochemistry, molecular biology 2012 Ionenkanal Ion-Channel Engineering: The monomeric porin OmpG as model OmpG ths Prof. Dr. Essen Lars-Oliver Essen, Lars-Oliver (Prof. Dr.) Membrane protein Protein Kristallographie doctoralThesis Membranprotein monograph Ion-Channel Engineering https://archiv.ub.uni-marburg.de/diss/z2012/0662/cover.png Große, Wolfgang Große Wolfgang Ion channel Membrane proteins like passive channels and pores, active transporters and receptors mediate the essential flow of information and matter across the permeability barrier generated by cell membranes. Due to their central role in the cell’s survival, membrane proteins form important targets for drug development by the pharmaceutical industry. Ion-Channel Engineering (ICE) utilizes chemical and biological methodologies to modify naturally occurring channels and pores to access and elucidate their function. Information derived from these membrane proteins can be applied to the design of complex systems as performed in bottom-up approaches of synthetic biology. This work exploits the potential of the monomeric porin OmpG to serve as a model for ICE as OmpG provides unique structural features amongst this class of proteins. This integral membrane protein from the outer membrane of the Gram-negative bacterium Escherichia coli shows a beta-barrel fold formed by 14 beta-strands and shows a large inner lumen of 12 · 15 Å diameter. The porin functions as a monomer and can be either extracted from membrane fractions or refolded from inclusion bodies using the rapid dilution technique. Two approaches were chosen to functionalize the OmpG porin: Functionalization of cysteines in the inner conductive pathway by S-alkylation or by the introduction of chemical alterations by N-peptides using the native chemical ligation (NCL) technique. As second step, copper (I)-catalyzed click chemistry was performed for diversification. Obtained hybrids were characterized by a combined approach using SDS-PAGE, fluorescence spectroscopy, mass spectrometry, black lipid membrane measurements (BLM measurements) and protein crystallography. All approaches yielded functionalized protein hybrids. Additionally, the first X-ray structure of a synthetically modified OmpG pore could be presented. The data including BLM-measurements mostly raised two challenges: reduction of flickering and attachment of the compounds to the pore for mobility reduction. To enforce a stable open state of the pore two deletion variants of the OmpG pore were prepared lacking the flexible loop6. Both variants could be refolded and formed functional channels with improved gating characteristics as indicated by BLM recordings. X-ray crystallographic data could be obtained for the variant bearing the larger deletion. The structural data revealed a novel, triclinic crystal form for the OmpG pore. Analysis of all observed crystal packings identified two hydrophobic interfaces, of which at least one is part of every structural assembly. Two strategies for the advanced attachment of a molecular bar through the middle of the OmpG conductive pathway were examined. One strategy focused on the direct introduction of the bar using the modification of cysteines by bifunctional labeling agents. The other strategy established the bar by later complexation of a metal ion using two independent, cystein-attached bipyridine ligands. The latter method appeared to have succeeded in establishing a molecular bar due to incorporated copper found by ICP-mass spectrometry and planar lipid bilayer recordings. In general, OmpG serves as an ideal template for ICE due to its unique structural features. This membrane protein could be optimized for sensing applications and functionalized through application of diverse methods. Modifizierung Protein crystallography Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (5259):680-685. Clark ED, Schwarz E and Rudolph R (1999) Inhibition of aggregation side reactions during in vitro protein folding. Amyloid 'Prions' and Other Protein Aggregates 309:217-236. Myers J, Grothaus G, Narayanan S and Onufriev A (2006) A simple clustering algorithm can be accurate enough for use in calculations of pKs in macromolecules. Proteins: Structure, Function, and Bioinformatics 63 (4):928-938. McCoy AJ, Grosse-Kunstleve RW, Storoni LC and Read RJ (2005) Likelihood-enhanced fast translation functions. Acta Crystallographica Section D 61 (4):458-464. Afonine PV, Grosse-Kunstleve RW and Adams PD (2005) The Phenix refinement framework. CCP4 Newsletter on protein crystallography 42:1-7. Tanner SD, Baranov VI and Bandura DR (2002) Reaction cells and collision cells for ICP-MS: a tutorial review. Spectrochimica Acta Part B: Atomic Spectroscopy 57 (9):1361-1452. Eswar N, John B, Mirkovic N, Fiser A, Ilyin VA, Pieper U, Stuart AC, Marti-Renom MA, Madhusudhan MS, Yerkovich B and Sali A (2003) Tools for comparative protein structure modeling and analysis. Nucleic Acids Research 31 (13):3375-3380. Sullivan BP, Salmon DJ and Meyer TJ (1978) Mixed phosphine 2,2'-bipyridine complexes of ruthenium. Inorganic Chemistry 17 (12):3334-3341. Hufnagel P, Schweiger U, Eckerskorn C and Oesterhelt D (1996) Electrospray ionization mass spectrometry of genetically and chemically modified bacteriorhodopsins. Analytical Biochemistry 243 (1):46-54. Bayley H, Cheley S, Harrington L and Syeda R (2009) Wrestling with Native Chemical Ligation. ACS Chemical Biology 4 (12):983-985. Gordon JC, Myers JB, Folta T, Shoja V, Heath LS and Onufriev A (2005) H++: a server for estimating pKas and adding missing hydrogens to macromolecules. Nucleic Acids Research 33:368-371. IDTDNA (2010) Calculation of T m for Oligonucleotide Duplexes. in http://eu.idtdna.com, 1-4. Middelberg APJ (2002) Preparative protein refolding. Trends in Biotechnology 20 (10):437-443. Koçer A, Walko M, Meijberg W and Feringa BL (2005) A Light-Actuated Nanovalve Derived from a Channel Protein. Science 309 (5735):755-758. Koçer A, Walko M, Bulten E, Halza E, Feringa BL and Meijberg W (2006) Rationally Designed Chemical Modulators Convert a Bacterial Channel Protein into a pH-Sensory Valve. Angewandte Chemie International Edition 45 (19):3126-3130. Winarski RP, Ederer DL, Kurmaev EZ, Shamin SN, Endo K, Ida T, Moewes A, Chang GS, Kim SY and Whang CN (1999) Soft X-ray fluorescence measurements of polyimide films. Thin Solid Films 357 (2):91-97. Mari SA, Köster S, Bippes CA, Yildiz Ö, Kühlbrandt W and Muller DJ (2010) pH-Induced Conformational Change of the β-Barrel-Forming Protein OmpG Reconstituted into Native E. coli Lipids. Journal of Molecular Biology 396 (3):610-616. Damaghi M, Bippes C, Köster S, Yildiz Ö, Mari SA, Kühlbrandt W and Muller DJ (2010) pH- Dependent Interactions Guide the Folding and Gate the Transmembrane Pore of the β-Barrel Membrane Protein OmpG. Journal of Molecular Biology 397 (4):878-882. Carey FA (2003) Organic chemistry (McGraw-Hill Companies, New York) 5th Ed: 1-1191. Mülhardt C (2006) Der Experimentator: Molekularbiologie/Genomics (Elsevier GmbH, München) 5. Ed: 1-298. Zava O, Zakeeruddin SM, Danelon C, Vogel H, Grätzel M and Dyson PJ (2009) A Cytotoxic Ruthenium Tris(Bipyridyl) Complex that Accumulates at Plasma Membranes. ChemBioChem 10 (11):1796-1800. Agre P (2004) Aquaporin Water Channels (Nobel Lecture). Angewandte Chemie International Edition 43 (33):4278-4290. Vagin AA, Steiner RA, Lebedev AA, Potterton L, McNicholas S, Long F and Murshudov GN (2004) REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallographica Section D 60 (12 Part 1):2184-2195. Clovis JS, Eckell A, Huisgen R and Sustmann R (1967) 1.3-Dipolare Cycloadditionen, XXV. Der Nachweis des freien Diphenylnitrilimins als Zwischenstufe bei Cycloadditionen. Chemische Berichte 100 (1):60-70. Carpino LA and Han GY (1972) 9-Fluorenylmethoxycarbonyl amino-protecting group. Journal of Organic Chemistry 37 (22):3404-3409. Hadfield A and Hajdu J (1993) A fast and portable microspectrophotometer for protein crystallography. Journal of applied crystallography 26 (6):839-842. Al-Momani La, Reiß P and Koert U (2005) A lipid dependence in the formation of twin ion channels. Biochemical and Biophysical Research Communications 328 (1):342-347. Wessel D and Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry 138 (1):141-143. Kushner SR (1978) An improved method for transformation of Escherichia coli with ColEl derived plasmids. Genetic Engineering:17-23. Takeda K, Sato H, Hino T, Kono M, Fukuda K, Sakurai I, Okada T and Kouyama T (1998) A novel three-dimensional crystal of bacteriorhodopsin obtained by successive fusion of the vesicular assemblies. Journal of Molecular Biology 283 (2):463-474. Tetko IV and Poda GI (2004) Application of ALOGPS 2.1 to Predict log D Distribution Coefficient for Pfizer Proprietary Compounds. Journal of Medicinal Chemistry 47 (23):5601- 5604. Tetko IV and Bruneau P (2004) Application of ALOGPS to predict 1-octanol/water distribution coefficients, logP, and logD, of AstraZeneca in-house database. Journal of Pharmaceutical Sciences 93 (12):3103-3110. Tetko IV and Tanchuk VY (2002) Application of Associative Neural Networks for Prediction of Lipophilicity in ALOGPS 2.1 Program. Journal of chemical information and computer sciences 42 (5):1136-1145. Bradford MM (1976) A Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry 72 (1-2):248-254. Pierotti RA (1976) A scaled particle theory of aqueous and nonaqueous solutions. Chemical Reviews 76 (6):717-726. Grant JA, Pickup BT, Sykes MJ, Kitchen CA and Nicholls A (2007) A simple formula for dielectric polarisation energies: The Sheffield Solvation Model. Chemical Physics Letters 441 (1- 3):163-166. Huber R (1989) A Structural Basis of Light Energy and Electron Transfer in Biology (Nobel Lecture). Angewandte Chemie International Edition 28 (7):848-869. Viswanadhan VN, Ghose AK, Revankar GR and Robins RK (1989) Atomic physicochemical parameters for three dimensional structure directed quantitative structure-activity relationships. 4. Additional parameters for hydrophobic and dispersive interactions and their application for an automated superposition of certain naturally occurring nucleoside antibiotics. Journal of chemical information and computer sciences 29 (3):163-172. Walker JE (1997) ATP Synthesis by Rotary Catalysis (Nobel Lecture). www.nobeprize.org:146 - 168. Außerdem gilt dem Personal der ESRF (Grenoble) ein großes Merci! Allen voran Tobias Klar, aber auch allen anderen, die mit uns zusammen die kleineren und größeren Probleme vor Ort gelöst haben. Gram HCJ and Friedlaender C (1884) Über die isolierte Färbung der Schizomyceten: in Schnitt- und Trockenpräparaten (Theodor Fischer's medicinischer Buchhandlung)185–189. Wieland T, Bokelmann E, Bauer L, Lang HU and Lau H (1953) Über Peptidsynthesen: 8. Mitteilung Bildung von S haltigen Peptiden durch intramolekulare Wanderung von Aminoacylresten. Justus Liebigs Annalen der Chemie 583 (1):129-149. Faham S and Bowie JU (2002) Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. Journal of Molecular Biology 316 (1):1- 6. Garrett TM, Koert U and Lehn J-m (1992) Binding cooperativity in the self-assembly of double stranded silver(I) trihelicates. Journal of Physical Organic Chemistry 5 (8):529-532. Grosse W (2008) Biohybride auf Basis des monomeren Porins OmpG. Diplomarbeit (Philipps- Universität, Marburg). Edsall JT and Wyman J (1958) Biophysical chemistry: Thermodynamics, electrostatics, and the biological significance of the properties of matter. (Academic Press, New York), Vol 1, 1-699. Mandel M and Higa A (1970) Calcium-dependent Bacteriophage DNA Infection. Journal of Molecular Biology 53 (1):159-162. Rodionova NA, Tatulian SA, Surrey T, Jaehnig F and Tamm LK (1995) Characterization of two membrane-bound forms of OmpA. Biochemistry 34 (6):1921-1929. Vinzenz X, Grosse W, Linne U, Meissner B and Essen L-O (2011) Chemical engineering of Mycobacterium tuberculosis dodecin hybrids. Chemical Communications 47 (39):11071-11073. Lübben W (2007) Chemoenzymatische Synthese von Hybridmembranproteinen basierend auf dem monomeren E. coli Porin OmpG. Diplomarbeit (Philipps-Universität, Marburg). Sánchez R and Šali A (2000) Comparative protein structure modeling (Humana Press, New Jersey) Mao Z-W, Heinemann FW, Liehr G and Eldik Rv (2001) Complex-formation reactions of Cu(ii) and Zn(ii) 2,2[prime or minute]-bipyridine and 1,10-phenanthroline complexes with bicarbonate. Identification of different carbonate coordination modes. Journal of the Chemical Society, Dalton Transactions (24):3652-3662. Dubendorff JW and Studier FW (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology 219 (1):45-59. Prilipov A, Phale PS, Van Gelder P, Rosenbusch JP and Koebnik R (1998) Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E-coli. FEMS Microbiology Letters 163 (1):65-72. Dubendorff JW and Studier FW (1991) Creation of a T7 autogene -cloning and expression of the gene for bacteriophage-T7 RNA-polymerase under control of its cognate promoter. Journal of Molecular Biology 219 (1):61-68. Pfeifer JR, Reiß P and Koert U (2006) Crown Ether–Gramicidin Hybrid Ion Channels: Dehydration-Assisted Ion Selectivity. Angewandte Chemie International Edition 45 (3):501-504. Krissinel E (2010) Crystal contacts as nature's docking solutions. Journal of Computational Chemistry 31 (1):133-143. Deisenhofer J, Epp O, Sinning I and Michel H (1995) Crystallographic refinement at 2.3 Å Resolution and Refined Model of the Photosynthetic Reaction Centre from Rhodopseudomonas viridis. Journal of Molecular Biology 246 (3):429-457. Danke für die konstante Arbeit, die den Fachbereich am Laufen hält. Insbesondere an unsere ganze Feinmechanik, Thomas Uderstadt und Werner Kirch. TCBgroup, Data Analysis in VMD, http://www.ks.uiuc.edu/Training/Tutorials/vmd/tutorial- html/node7.html, (2011). Krissinel E and Henrick K (2005) Detection of Protein Assemblies in Crystals. Computational Life Sciences, Lecture Notes in Computer Science, eds R. Berthold M, Glen R, Diederichs K, Kohlbacher O and Fischer I (Springer, Berlin / Heidelberg), Vol 3695, 163-174. Susanne J., Julius L., Anne N., Kathrin P., Anastasia S., Philipp S., Julia S. und Xenia V. inklusive des Start-Teams Harm O., Florian P., Julia P. (ja, hier bleibst du P und bist nicht S), Bodo S., unseren Bacheloretten und Master-Studenten Silke v. H., Vitali K., Michael K., Christin S., Dennis W. und zu guter Letzt den wenigen, die ich nur in ihren letzten Tagen erleben durfte Sevgi C., Sabine G., Wolger L., Holger W. und Ying Z.. Ich fand wir hatten eine super Zeit mit viel Spaß zusammen, der mit Einigen über ein reines Arbeits-Verhältnis weit hinaus ging und haben viele Probleme gut zusammen gemeistert. Unsere AK-Abende sind die Besten! Die Service-Einrichtungen des Fachbereichs sollen an dieser Stelle auch nicht unerwähnt bleiben. Kuntal B, Aparoy P and Reddanna P (2010) EasyModeller: A graphical interface to MODELLER. BMC Research Notes 3 (1):226. Ein großer Dank gilt auch den vielen Sekretariaten des Fachbereichs Chemie, die immer hilfsbereit bei allen Problemen unterstützt haben; Insbesondere natürlich unserer Rosi Roller-Müller. Ein großer Dank gilt auch der Massenspektrometrischen Abteilung! Uwe Linne und Natalia Fritzler haben durch ihr konstantes Interesse und ein großes Gespür für die Parameter die nicht trivialen MS- Nachweise von OmpG erst ermöglicht und hatten immer ein offenes Ohr und ausreichend Zeit. Shigekawa K and Dower WJ (1988) Electroporation of eukaryotes and prokaryotes: a general approach to the introduction of macromolecules into cells. Biotechniques 6 (8):742-751. Tetko IV, Tanchuk VY, Kasheva TN and Villa AEP (2001) Estimation of Aqueous Solubility of Chemical Compounds Using E-State Indices. Journal of chemical information and computer sciences 41 (6):1488-1493. Jakalian A, Jack DB and Bayly CI (2002) Fast, efficient generation of high-quality atomic charges. AM1-BCC model: II. Parameterization and validation. Journal of Computational Chemistry 23 (16):1623-1641. 6. Literatur 155 Back K (2010) Funktionelle Charakterisierung von mVDAC1-Basis für Ion Channel Engineering. Diplomarbeit (Philipps-Universität, Marburg). Danke euch allen! Bis bald, alles Liebe, Wolfgang OmpG/1-18 nach SPPS 18er-Peptid nativ Boc-Glu(tBu)-Glu(tBu)-Arg(Pbf)-Asn(Trt)-Asp(tBu)- Trp(Boc)-His(Trt)-Phe-Asn(Trt)-Ile-Gly-Ala-Met- Tyr(tBu)-Glu(tBu)-Ile-Glu(tBu)-Gly-OH R= OmpG/1-18/ 13 Boc-Glu(tBu)-Glu(tBu)-Arg(Pbf)-Asn(Trt)-Asp(tBu)- Trp(Boc)-His(Trt)-Phe-Asn(Trt)-Ile-Gly-Ala- - Tyr(tBu)-Glu(tBu)-Ile-Glu(tBu)-Gly-OH R= OmpG/1-18 entschützt und aktiviert OmpG/1-18 Dower WJ, Miller JF and Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Research 16 (13):6127-6145. Krissinel E and Henrick K (2007) Inference of Macromolecular Assemblies from Crystalline State. Journal of Molecular Biology 372 (3):774-797. Demmers JAA, van Dalen A, de Kruijff B, Heck AJR and Killian JA (2003) Interaction of the K+ channel KcsA with membrane phospholipids as studied by ESI mass spectrometry. FEBS Letters 541 (1-3):28-32. Karas M and Hillenkamp F (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Analytical Chemistry 60 (20):2299-2301. Lehninger AL, Nelson DL and Cox MM (2005) Lehninger principles of biochemistry (WH Freeman + Co) fourth Ed: 1-1119. Wlodek S, Skillman AG and Nicholls A (2010) Ligand Entropy in Gas-Phase, Upon Solvation and Protein Complexation. Fast Estimation with Quasi-Newton Hessian. Journal of Chemical Theory and Computation 6 (7):2140-2152. Banghart M, Borges K, Isacoff E, Trauner D and Kramer RH (2004) Light-activated ion channels for remote control of neuronal firing. Nature Neuroscience 7 (12):1381-1386. Wadsten P, Wöhri AB, Snijder A, Katona G, Gardiner AT, Cogdell RJ, Neutze R and Engström S (2006) Lipidic Sponge Phase Crystallization of Membrane Proteins. Journal of Molecular Biology 364 (1):44-53. Heimburg T (2010) Lipid ion channels. Biophysical Chemistry 150 (1-3):2-22. Essen L-O, Siegert R, Lehmann WD and Oesterhelt D (1998) Lipid patches in membrane protein oligomers: Crystal structure of the bacteriorhodopsin-lipid complex. Proceedings of the National Academy of Sciences of the United States of America 95 (20):11673-11678. Bertani G (2004) Lysogeny at mid-twentieth century: P1, P2, and other experimental, systems. Journal of Bacteriology 186 (3):595-600. Inverarity IA and Hulme AN (2007) Marked small molecule libraries: a truncated approach to molecular probe design. Organic + Biomolecular Chemistry 5 (4):636-643. Johansson LC, Wöhri AB, Katona G, Engström S and Neutze R (2009) Membrane protein crystallization from lipidic phases. Current Opinion in Structural Biology 19 (4):372-378. Halgren TA (1996) Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94. Journal of Computational Chemistry 17 (5-6):490-519. Halgren TA (1996) Merck molecular force field. III. Molecular geometries and vibrational frequencies for MMFF94. Journal of Computational Chemistry 17 (5-6):553-586. Halgren TA (1996) Merck molecular force field. II. MMFF94 van der Waals and electrostatic parameters for intermolecular interactions. Journal of Computational Chemistry 17 (5-6):520- 552. Halgren TA and Nachbar RB (1996) Merck molecular force field. IV. conformational energies and geometries for MMFF94. Journal of Computational Chemistry 17 (5-6):587-615. Halgren TA (1996) Merck molecular force field. V. Extension of MMFF94 using experimental data, additional computational data, and empirical rules. Journal of Computational Chemistry 17 (5-6):616-641. Iwata S (2003) Methods and results in crystallization of membrane proteins (International University Line)1-355. Munk K (2001) Mikrobiologie (Spektrum Akademischer Verlag, Heidelberg)1-1242. Halgren TA (1999) MMFF VII. Characterization of MMFF94, MMFF94s, and other widely available force fields for conformational energies and for intermolecular-interaction energies and geometries. Journal of Computational Chemistry 20 (7):730-748. Halgren TA (1999) MMFF VI. MMFF94s option for energy minimization studies. Journal of Computational Chemistry 20 (7):720-729. Fiser A and Šali A (2003) Modeller: Generation and Refinement of Homology-Based Protein Structure Models. Methods in Enzymology, eds Charles W. Carter, Jr. and Robert MS (Academic Press), Vol Volume 374, 461-491. Vagin A and Teplyakov A (1997) MOLREP: an Automated Program for Molecular Replacement. Journal of applied crystallography 30 (6):1022-1025. Ohne Euch wäre diese Arbeit niemals möglich gewesen und ich freue mich auf ein paar weniger arbeitsreiche Tage in Eurer Mitte. Schomaker V and Trueblood KN (1968) On the rigid-body motion of molecules in crystals. Acta Crystallographica Section B 24 (1):63-76. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC and Zwart PH (2010) PHENIX: a comprehensive Python- 6. Literatur 157 based system for macromolecular structure solution. Acta Crystallographica Section D 66 (2):213-221. Bashford D and Karplus M (1990) pKa's of ionizable groups in proteins: atomic detail from a continuum electrostatic model. Biochemistry 29 (44):10219-10225. MacKinnon R (2004) Potassium Channels and the Atomic Basis of Selective Ion Conduction (Nobel Lecture). Angewandte Chemie International Edition 43 (33):4265-4277. Tetko IV, Tanchuk VY and Villa AEP (2001) Prediction of n-Octanol/Water Partition Coefficients from PHYSPROP Database Using Artificial Neural Networks and E-State Indices. Journal of chemical information and computer sciences 41 (5):1407-1421. Perkins DN, Pappin DJC, Creasy DM and Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20 (18):3551-3567. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD and Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. The proteomics protocols handbook:571-607. le Coutre J, Whitelegge JP, Gross A, Turk E, Wright EM, Kaback HR and Faull KF (2000) Proteomics on Full-Length Membrane Proteins Using Mass Spectrometry. Biochemistry 39 (15):4237-4242. DeLano WL (2011) PyMOL Molecular Graphics System (Schrödinger LLC), 1.4. Bennett J and Scott KJ (1971) Quantitative staining of Fraction I protein in polyacrylamide gels using coomassie brilliant blue. Analytical Biochemistry 43 (1):173-182. Pappin DJC, Hojrup P and Bleasby AJ (1993) Rapid identification of proteins by peptide-mass fingerprinting. Current Biology 3 (6):327-332. Brückner R (2004) Reaktionsmechanismen (Spektrum Alademischer Verlag GmbH, Heidelberg) third Ed: 1-863. Murshudov GN, Vagin AA and Dodson EJ (1997) Refinement of Macromolecular Structures by the Maximum-Likelihood Method. Acta Crystallographica Section D 53 (3):240-255. Schimper AFW (1883) Über die Entwickelung der Chlorophyllkörner und Farbkörper. Botanische Zeitung 41 (7):105 -160. Schlosser für die Zusammenarbeit in der Synthese der N-Peptide und Britta Meißner für das Überlassen ihres Dodecin-Datensatzes. Garrett TM, Koert U, Lehn JM, Rigault A, Meyer D and Fischer J (1990) Self-assembly of silver (I) helicates. Chemical Communications (7):557-558. Moriguchi I, Hirono S, Liu Q, Nakagome I and Matsushita Y (1992) Simple method of calculating octanol/water partition coefficient. Chemical and Pharmaceutical Bulletin 40 (1):127- 130. Merrifield RB (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society 85 (14):2149-2154. Becker T, Vögtle FN, Stojanovski D and Meisinger C (2008) Sorting and assembly of mitochondrial outer membrane proteins. Biochimica et Biophysica Acta (BBA) -Bioenergetics 1777 (7-8):557-563. Vinzenz X (2010) Sphärische Dodecin-Biohybride als Basis für schaltbare Katalysatoren. Diplomarbeit (Philipps-Universität, Marburg). McQuarrie D (1976) Statistical Mechanics. (New York: Harper + Row). Koebnik R, Locher KP and Van Gelder P (2000) Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Molecular Microbiology 37 (2):239-253. Chang G, Spencer RH, Lee AT, Barclay MT and Rees DC (1998) Structure of the MscL Homolog from Mycobacterium tuberculosis: A Gated Mechanosensitive Ion Channel. Science 282 (5397):2220-2226. Mertins B (2011) Strukturelle und funktionelle Charakterisierung von mVDAC1-Biohybriden. Diplomarbeit (Philipps-Universität, Marburg). Cebi M (2008) Synthese und Modifikation von Hybridionenkanälen. Dissertation (Philipps- Universität, Marburg). Hirsch B (2009) Synthese von Bis(iodacetamiden) als Bausteine zur Porenzweipunktanknüpfung. Diplomarbeit (Philipps-Universität, Marburg). CollaborativeComputationalProject4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallographica Section D 50 (5):760-763. Albrecht AG, Landmann H, Nette D, Burghaus O, Peuckert F, Seubert A, Miethke M and Marahiel MA (2011) The Frataxin Homologue Fra Plays a Key Role in Intracellular Iron Channeling in Bacillus subtilis. ChemBioChem 12 (13):2052-2061. Skou JC (1998) The Identification of the Sodium–Potassium Pump (Nobel Lecture). Angewandte Chemie International Edition 37 (17):2320-2328. Wallin IE (1923) The Mitochondria Problem. The American Naturalist 57 (650):255-261. Deisenhofer J and Michel H (1989) The Photosynthetic Reaction Center from the Purple Bacterium Rhodopseudomonas viridis (Nobel Lecture). Angewandte Chemie International Edition 28 (7):829-847. Schulz GE (2002) The structure of bacterial outer membrane proteins. Biochimica et Biophysica Acta (BBA) -Biomembranes 1565 (2):308-317. Weiss MS, Wacker T, Weckesser J, Weite W and Schulz GE (1990) The three-dimensional structure of porin from Rhodobacter capsulatus at 3 Å resolution. FEBS Letters 267 (2):268-272. Wimley WC (2003) The versatile β-barrel membrane protein. Current Opinion in Structural Biology 13 (4):404-411. Morrison DA (1977) Transformation in Escherichia coli: Cryogenic Preservation of Competent Cells. Journal of Bacteriology 132 (1):349-351. Rose F (1833) Ueber die Verbindungen des Eiweiss mit Metalloxyden. Annalen der Physik 104 (5):132-142. Tetko I, Gasteiger J, Todeschini R, Mauri A, Livingstone D, Ertl P, Palyulin V, Radchenko E, Zefirov N, Makarenko A, Tanchuk V and Prokopenko V (2005) Virtual Computational Chemistry Laboratory – Design and Description. Journal of Computer-Aided Molecular Design 19 (6):453-463. Mestroni G, Alessio E, Sava G, Pacor S, Coluccia M and Boccarelli A (1994) Water-Soluble Ruthenium(III)-Dimethyl Sulfoxide Complexes: Chemical Behaviour and Pharmaceutical Properties. Metal-Based Drugs 1 (1):41-63. Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1984) X-ray structure analysis of a membrane protein complex: Electron density map at 3 Å resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis. Journal of Molecular Biology 180 (2):385-398. Anbazhagan V, Qu J, Kleinschmidt JH and Marsh D (2008) Incorporation of Outer Membrane Protein OmpG in Lipid Membranes: Protein−lipid Interactions and β-Barrel Orientation. Biochemistry 47 (23):6189-6198. Koenig T, Menze BH, Kirchner M, Monigatti F, Parker KC, Patterson T, Steen JJ, Hamprecht FA and Steen H (2008) Robust Prediction of the MASCOT Score for an Improved Quality Assessment in Mass Spectrometric Proteomics. Journal of Proteome Research 7 (9):3708-3717. Winn MD, Murshudov GN and Papiz MZ (2003) Macromolecular TLS Refinement in REFMAC at Moderate Resolutions. Methods in Enzymology, eds Charles W. Carter, Jr. and Robert MS (Academic Press), Vol Volume 374, 300-321. Rozenski J, Peptide Mass Calculator v3.2, http://rna.rega.kuleuven.ac.be/masspec/pepcalc.htm. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC and Read RJ (2007) Phaser crystallographic software. Journal of applied crystallography 40 (4):658-674. Potterton E, Briggs P, Turkenburg M and Dodson E (2003) A graphical user interface to the CCP4 program suite. Acta Crystallographica Section D 59 (7):1131-1137. Emsley P and Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallographica Section D 60 (12 Part 1):2126-2132. Kocer A, Walko M and Feringa BL (2007) Synthesis and utilization of reversible and irreversible light-activated nanovalves derived from the channel protein MscL. Nature Protocols 2 (6):1426- 1437. Mereschkowsky C (1905) Über Natur und Ursprung der Chromatophoren im Pflanzenreiche. Biologisches Centralblatt (25):593–604. Krissinel E and Henrick K, Protein interfaces, surfaces and assemblies service PISA, http://www.ebi.ac.uk/pdbe/prot_int/pistart.html, (2011). Michel H (1983) Crystallization of membrane proteins. Trends in Biochemical Sciences 8 (2):56- 59. Friehs K (2004) Plasmid copy number and plasmid stability 182. Novagen (1998) pET-20b(+) Vector. in http://www.merck-chemicals.de, (Merck KGaA), 1-2. Banghart MR, Mourot A, Fortin DL, Yao JZ, Kramer RH and Trauner D (2009) Photochromic Blockers of Voltage-Gated Potassium Channels. Angewandte Chemie International Edition 48 (48):9097-9101. Volgraf M, Gorostiza P, Numano R, Kramer RH, Isacoff EY and Trauner D (2006) Allosteric control of an ionotropic glutamate receptor with an optical switch. Nature Chemical Biology 2 (1):47-52. Strong M, Sawaya MR, Wang S, Phillips M, Cascio D and Eisenberg D (2006) Toward the structural genomics of complexes: Crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 103 (21):8060-8065. Gorostiza P, Volgraf M, Numano R, Szobota S, Trauner D and Isacoff EY (2007) Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor. Proceedings of the National Academy of Sciences of the United States of America 104 (26):10865-10870. Liang B and Tamm LK (2007) Structure of outer membrane protein G by solution NMR spectroscopy. Proceedings of the National Academy of Sciences of the United States of America 104 (41):16140-16145. Landau EM and Rosenbusch JP (1996) Lipidic cubic phases: A novel concept for the crystallization of membrane proteins. Proceedings of the National Academy of Sciences of the United States of America 93 (25):14532-14535. Wunderlich B, Leirer C, Idzko AL, Keyser UF, Wixforth A, Myles VM, Heimburg T and Schneider MF (2009) Phase-State Dependent Current Fluctuations in Pure Lipid Membranes. Biophysical Journal 96 (11):4592-4597. Moriarty NW, Grosse-Kunstleve RW and Adams PD (2009) electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallographica Section D 65 (10):1074-1080. Fortin DL, Banghart MR, Dunn TW, Borges K, Wagenaar DA, Gaudry Q, Karakossian MH, Otis TS, Kristan WB, Trauner D and Kramer RH (2008) Photochemical control of endogenous ion channels and cellular excitability. Nature Methods 5 (4):331-338. Miller JF, Dower WJ and Tompkins LS (1988) High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA. Proceedings of the National Academy of Sciences of the United States of America 85 (3):856-860. Chen VB, Arendall WB, III, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS and Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica Section D 66 (1):12-21. Komarov AG, Linn KM, Devereaux JJ and Valiyaveetil FI (2009) Modular Strategy for the Semisynthesis of a K+ Channel: Investigating Interactions of the Pore Helix. ACS Chemical Biology 4 (12):1029-1038. Valiyaveetil FI, Sekedat M, Muir TW and MacKinnon R (2004) Semisynthesis of a Functional K+ Channel. Angewandte Chemie International Edition 43 (19):2504-2507. Eckhardt B, Grosse W, Essen L-O and Geyer A (2010) Structural characterization of a β-turn mimic within a protein–protein interface. Proceedings of the National Academy of Sciences of the United States of America 107 (43):18336-18341. Lehn JM, Rigault A, Siegel J, Harrowfield J, Chevrier B and Moras D (1987) Spontaneous assembly of double-stranded helicates from oligobipyridine ligands and copper(I) cations: structure of an inorganic double helix. Proceedings of the National Academy of Sciences of the United States of America 84 (9):2565-2569. Guengerich FP (2011) Thematic Minireview Series: Biological Applications of Mass Spectrometry. Journal of Biological Chemistry 286 (29):25417. Dhakshnamoorthy B, Raychaudhury S, Blachowicz L and Roux B (2010) Cation-selective Pathway of OmpF Porin Revealed by Anomalous X-ray Diffraction. Journal of Molecular Biology 396 (2):293-300. Valiyaveetil FI, Sekedat M, MacKinnon R and Muir TW (2004) Glycine as a d-amino acid surrogate in the K+-selectivity filter. Proceedings of the National Academy of Sciences of the United States of America 101 (49):17045-17049. Neher E (1992) Ion channels for communication between and within cells (Nobel Lecture). Geist AB, Dongarra J, Jiang W, Manchek R and Sunderam VS (1994) PVM: Parallel Virtual Machine: a users' guide and tutorial for network parallel computing. Terwilliger TC, Grosse-Kunstleve RW, Afonine PV, Moriarty NW, Zwart PH, Hung L-W, Read RJ and Adams PD (2008) Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallographica Section D 64 (1):61-69. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ and Klenk DC (1985) Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150 (1):76-85. Humphrey W, Dalke A and Schulten K (1996) VMD: Visual molecular dynamics. Journal of Molecular Graphics 14 (1):33-38. Feliciello I and Chinali G (1993) A Modified Alkaline Lysis Method for the Preparation of Highly Purified Plasmid DNA from Escherichia coli. Analytical Biochemistry 212 (2):394-401. Šali A and Blundell TL (1993) Comparative Protein Modelling by Satisfaction of Spatial Restraints. Journal of Molecular Biology 234 (3):779-815. Gallaher J, Wodzińska K, Heimburg T and Bier M (2010) Ion-channel-like behavior in lipid bilayer membranes at the melting transition. Physical Review E 81 (6):061925. OmpG 2012-10-09 urn:nbn:de:hebis:04-z2012-06626 Kristallographie Ion-Channel Engineering Ion-Channel Engineering: Das monomere Porin OmpG als Modell Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg German Membranproteine wie passive Kanäle und Poren, aktive Transporter und Rezeptoren regulieren den essentiellen Fluss von Informationen und Substanzen über durch Zellmembranen gebildete Permeabilitäts-Barrieren. Aufgrund dieser zentralen Rolle stehen Membranproteine als Angriffspunkt für Wirkstoffe im Fokus des Interesses der pharmazeutischen Industrie. Die Technik des Ion-Channel Engineering (ICE) modifiziert natürliche Kanäle durch chemische und biologische Werkzeuge, um Einblick in die Funktionsweise von Kanälen und Poren zu erhalten. Die erhaltenen Informationen bilden des Weiteren eine Basis für die Verwendung von Ionenkanälen als Bausteine für die Gestaltung komplexer biologischer Systeme, wie sie durch die Synthetische Biologie erforscht werden. In der vorliegenden Arbeit wurden die einzigartigen strukturellen Eigenschaften des monomeren Porins OmpG für die Nutzung als Modellsystem im Feld des ICE untersucht. Das integrale Membranprotein aus der äußeren Membran des Gram-negativen Bakteriums Escherichia coli bildet ein beta-barrel aus 14 beta-Strängen mit einem großen Innendurchmesser von 12 · 15 Å, das als Monomer in Membranen aktiv ist. Das Membranprotein wurde sowohl aus der Membran extrahiert als auch aus Inclusion Bodies mittels rapid dilution rückgefaltet eingesetzt. Zur Funktionalisierung von OmpG wurden zwei Ansätze gewählt, die Funktionalisierung von Cysteinen im zentralen Ionenleitweg des Porins mittels S-Alkylierung und die Funktionalisierung der Pore mittels synthetisierter N-Peptide über Native Chemische Ligation (NCL). Ein zweiter Schritt zur weiteren Modifikation erfolgte durch Cu(I)-katalysierte Click-Chemie. Erzeugte Biohybrid-Poren wurden mit einer Kombination aus SDS-PAGE, Fluoreszenz-Spektroskopie, Massenspektrometrie, Black Lipid Membrane-Messungen (BLM-Messungen) und Proteinkristallisation ausführlich charakterisiert. Beide Modifikationsrouten führten zu funktionellen Biohybriden und die erste Röntgenstruktur eines kovalent funktionalisierten OmpG-Membranproteins wurde erhalten. Die Daten zeigten, dass eine Optimierung der Eigenschaften der Pore für BLM-Messungen und der Aufhängung von Funktionalisierungen im Inneren der Pore notwendig waren. Zur Stabilisierung des Offen-Zustands der Pore wurden zwei verschiedene Deletionen des flexiblen loop6 vorgenommen und charakterisiert. Beide Varianten des Membranproteins waren faltungs-kompetent und zeigten verbesserte Eigenschaften in den BLM-Messungen. Für die Variante des Porins mit der maximalen Verkürzung des loops konnte eine Röntgenstruktur in einer neuen, triklinen Kristallform erhalten werden. Eine Analyse der Kristallpackungen der OmpG-Strukturen ergab zwei hydrophobe Interfaces, von denen mindestens eines in allen verfügbaren Kristallpackungen des Membranproteins OmpG zu beobachten ist. Die Optimierung der Funktionalisierungsaufhängung wurde durch das Einführen einer molekularen Strebe quer durch den Ionenleitweg des Membranproteins durchgeführt. Hierfür wurden zwei Strategien getestet, das direkte Einführen einer Strebe mittels bifunktioneller Verbindungen an Cysteinen und die Verstrebung von zwei unabhängigen Modifikationen initiiert durch Komplexierung eines Metallions. Für den Erfolg letzterer Strategie liegen Indizien durch den Nachweis gebundenen Kupfers mittels ICP-Massenspektrometrie und BLM-Messungen vor. OmpG bietet aufgrund seiner einzigartigen Struktur ein vielversprechendes Templat für das ICE. Das Membranprotein konnte in dieser Arbeit durch Deletion von loop6 für die Anwendung optimiert und verschiedene Strategien zur Erzeugung funktionalisierter Poren konnten erfolgreich etabliert werden. Fachbereich Chemie Philipps-Universität Marburg