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Biochemical characterization and crystallization of porin from Rhodopseudomonas blastica

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Abstract

Oligomeric porin of the phototrophic bacterium Rhodopseudomonas blastica DSM 2131 was obtained from cell envelopes by differential temperature extraction in the presence of detergent and salt. The isolated porin exhibited strong porin activity after reconstitution into lipid bilayer membranes. The effective channel diameter for the trimer was estimated as 1.5 nm from single channel conductance measurements in the presence of 1 M KCl. Moderate cation-selectivity was observed. Oligomeric porin migrated as a single band (apparent molecular weight 81 kDa) on sodium dodecyl sulfate polyacrylamide gelelectrophoresis when solubilized below 70 °C. The oligomers were converted into monomers on heating to 70 °C or above forming two bands with apparent molecular weight of 36 kDa and 35 kDa. The oligomer was not sensitive to EDTA. Its molecular weight was determined to be 119.3 kDa by analytical ultracentrifugation. The isoelectric point was 5.7. Circular dichroism data indicated a high content of β-sheet structure. Gasphase sequencing of the N-terminal residues revealed the sequence: NH2-Glu-Ile-Ser-Leu-Asn-Gly-Tyr-Gly-Arg-Phe. Crystals with a maximal side length of 300 μm and diffracting to 0.32 nm resolution were obtained with the porin oligomer in the presence of C8E4 and 1,2,3-heptanetriol by using the vapor phase equilibration technique.

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Abbreviations

C8E4:

n-octyl tetraoxyethylene

Mr :

apparent molecular weight

Octyl-POE:

n-octyl polyoxyethylene

LDAO:

N,N-dimethyl dodecyl aminoxide

LPS:

lipopolysaccharide

PAGE:

polyacrylamide gel-electrophoresis

PEG:

polyethylene glycol

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Authors and Affiliations

  1. Institut für Biologie II, Mikrobiologie, Schänzlestrasse 1, W-7800, Freiburg i.Br., Germany

    S. Butz & J. Weckesser

  2. Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, W-8700, Würzburg, Germany

    R. Benz

  3. Institut für Biophysik und Strahlenbiologie, Albertstrasse 23, W-7800, Freiburg i.Br., Germany

    T. Wacker & W. Welte

  4. Abteilung Biophysikalische Chemie, Biozentrum der Universität, Klingelbergstrasse 70, CH-4056, Basel, Switzerland

    A. Lustig

  5. Lehrstuhl für Mikrobiologie, Erwin-Schrödingerstrasse, W-6750, Kaiserslautern, Germany

    R. Plapp

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  1. S. Butz
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  2. R. Benz
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  3. T. Wacker
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  4. W. Welte
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  5. A. Lustig
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  6. R. Plapp
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  7. J. Weckesser
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Butz, S., Benz, R., Wacker, T. et al. Biochemical characterization and crystallization of porin from Rhodopseudomonas blastica . Arch. Microbiol. 159, 301–307 (1993). https://doi.org/10.1007/BF00290911

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  • DOI: https://doi.org/10.1007/BF00290911

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