The compatible solute-binding protein OpuAC from Bacillus subtilis: ligand-binding, site directed mutagenesis and crystallographic studies
Smits SH, Höing M, Lecher J, Jebbar M, Schmitt L, Bremer E.
Institute of Biochemistry, Heinrich Heine University Duesseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany; Laboratory for Microbiology, Department of Biology, Philipps University Marburg, Karl-von-Frisch Str., 35032 Marburg, Germany; Departement Osmoregulation chez les Bacteries, Universite de Rennes 1, UMR-CNRS 6026, Rennes, Frances.
In the soil bacterium Bacillus subtilis, five transport systems work in concert to mediate the import of various compatible solutes that counteract the deleterious effects of increases in the osmolarity of the environment. Among these five systems, the ABC transporter OpuA, which catalyses the import of glycine betaine and proline betaine has been studied in detail in the past. Here, we demonstrate that OpuA is capable of importing the sulfobetaine dimethylsulfonioacetate (DMSA). Since OpuA is a classic ABC importer that relies on a substrate-binding protein priming the transporter with specificity and selectivity, we analyzed the OpuA-binding protein, OpuAC, by structural and mutational means with respect to DMSA binding. The determined crystal structures of OpuAC in complex with DMSA at 2.8 A resolution and a detailed mutational analysis of these residues revealed a hierarchy within the amino acids participating in substrate binding. This finding is different to other binding proteins that recognize compatible solutes. Furthermore important principles that enable OpuAC to specifically bind various compatible solutes were uncovered.
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Chem competent Cells
Hier das Protokol zur Erstellung chemisch kompetenter E. coli Zellen nach Hanahan.
Protocol
Hanahan Competent Cell Protocol
Leslie Vosshall
**USE STERILE TECHNIQUE AT ALL TIMES
**AFTER STEP 6, WORK ON ICE IN THE COLD ROOM TO INCREASE THE QUALITY
OF THE FINAL CELL PREPARATION.
**PROTOCOL PRODUCES ABOUT 160 X 250ul ALIQUOTS OF COMPETENT CELLS.
PLACE SUFFICIENT 1.7ml MICROCENTRIFUGE TUBES AT –80 DEGREES C. AT THE
BEGINNING OF THE DAY FOR USE IN STEP #15.
- Streak out frozen stock of bacteria on an LB (+ antibiotics) plate. Grow overnight at 37 degrees C.
- Inoculate a single colony into a 250 ml flask containing:
- Grow overnight in a shaker at 37 degrees C.
- The next day set up two 2 liter flasks containing 250 ml 2XYT. Into each flask, pipet 2.5 ml of overnight culture.
- Grow with shaking until culture reaches an OD600 of ~0.5.
- Set up two centrifuge bottles on wet ice and transfer culture to bottles by pouring.
- Pellet bacteria by spinning in Sorvall centrifuge in GSA rotor; 5000 r.p.m., 4 degrees C.,10 minutes.
- Working in the cold room, with cells on ice as much as possible: Pour out supernatant, then use pipet to remove all residual fluid.
- Resuspend each pellet gently in 83 ml RF1. Use 10 ml pipet to gently pipet up and down until all clumps have been resuspended.
- Incubate on wet ice in cold room for 1 hour.
- Pellet bacteria by spinning in Sorvall centrifuge in GSA rotor; 5000 r.p.m., 4 degrees C., 10 minutes.
- Working in the cold room, with cells on ice as much as possible: Pour out supernatant, then use pipet to remove all residual fluid.
- Resuspend each pellet gently in 20 ml of RF2. Use 10 ml pipet to gently pipet up and down until all clumps have been resuspended.
- Incubate on wet ice in cold room for 15 minutes.
- Take microcentrifuge tubes out of –80 degree C freezer and at least 160 tubes with open lids in an ice bucket containing wet ice.
Dispense 100 ul of cell suspension into each tube. If possible, work with a second person, one person dispensing the cells,
and the second person sealing tubes as they are filled. Once tubes are sealed, drop them in a Dewar flask containing liquid nitrogen.
Once all 40 ml of competent cells are dispensed into microcentrifuge tubes, freeze all remaining tubes in liquid nitrogen.
Collect tubes and store in a paper freezer box with no dividers. - Measure the efficiency of the competent cells by transforming a standard amount of commercial plasmid. Express efficiency
as colony forming units / microgram of DNA cfu/ug. If you transform 0.1 ng of plasmid DNA and obtain 500 colonies, the efficiency
of the cells is 5 x 106 cfu/ug.
SOLUTIONS
RF1
FW PER LITER
100mM Rubidium Chloride 120.9 12.1 g
50mM Manganese Chloride 197.9 9.895 g
30mM Potassium Acetate 98.14 2.944 g
10mM Calcium Chloride 147.0 1.47 g
15% w/v Glycerol 150 g
Adjust pH to 5.8 and bring volume to 1 liter.
Sterilize by filtration.
RF2
FW PER 500ML
10mM MOPS 209.3 1.05 g
10mM Rubidium Chloride 120.9 0.6 g
75mM Calcium Chloride 147.0 5.51 g
15% w/v Glycerol 75 g
Bring volume to 500 ml.
Sterilize by filtration.
YT medium (2x)
PER LITER
Bacto tryptone 16g
Bacto yeast extract 10g
NaCl 5g
Bring volume to 1 liter.
Sterilize by autoclave.
Funktionale Charakterisierung des ABC-Transporters LmrA aus L. lactis
eingereicht von Justin Schmitz aus Hilden
Erstgutachter: Prof. Dr. Lutz Schmitt; Institut für Biochemie, Abteilung für Membrantransport; Heinrich-Heine Universität, Düsseldorf
Zweitgutachter: PD Dr. Ulrich Schulte; Institut für Biochemie, Arbeitsgruppe Mitochondrienbiogenese; Heinrich-Heine Universität, Düsseldorf
Zusammenfassung
ABC-Transporter sind primär aktive Transporter, deren Aufgabe in der Regel darin besteht, unter dem Transport unterschiedlicher Substrate über biologische Membranen zu kanalisieren. Für die ATP-Bindung und Hydrolyse liegen überzeugende Modelle vor die durch strukturelle und funktionelle Daten gestützt werden. Der Transport hingegen ist weitgehend unverstanden. In der vorliegenden Arbeit sollte daher ein System etabliert werden, um der Substratbindungsstelle des MDR-Exporters LmrA aus L. lactis zu charakterisieren, wodurch tiefergehende Einblicke in den Transportprozess möglich wäre.
Die vorliegende Arbeit unterteilt sich in zwei wesentliche Bereiche. Zum Einen die Homologiemodellierung von LmrA auf Basis der Struktur des ABC-Transporters Sav1866. Zum Anderen die Etablierung der Expression von LmrA, welches mit unnatürlichen Aminosäuren markiert ist. Hierzu wurde ein von der Gruppe von Professor Schultz (Scripps Research Institute, La Jolla, California USA) entwickeltes System zur Erweiterung des genetischen Kodes verwendet.
Auf Grundlage der signifikanten Homologie der beiden ABC-Transporter LmrA und Sav1866 wurde ein Homologiemodell für LmrA erstellt, welches biochemische Daten bezüglich Topologie und Funktion schlüssig erklären kann. Die in der Literatur wichtigsten Aminosäuren für die Transportfunktion von LmrA, aber auch seines humanen Homologen P-gp (ABCB1), liegen in der Transmembranhelix 6. Die TM6 ragt im Homologiemodell in den Hohlraum zwischen den beiden Hälften des Transporters. Diese exponierte Lage unterstreicht die Bedeutung für den Transportprozess von LmrA. Auf Grund dessen wurde die Suche nach der Substratbindungsstelle von LmrA in der TM6 begonnen. Hierzu wurden entlang der gesamten Helix 16 Positionen für Mutanten gewählt, die helfen sollten die Substratbindungsstelle mit spektroskopischen Verfahren zu lokalisieren.
Für die Untersuchungen der Substratbindungsstelle von LmrA sollte das Protein spezifisch mit unnatürlichen Aminosäuren modifiziert werden. Um dies zu realisieren, wurde die Expression von LmrA in E. coli etabliert, da nur für diesen Organismus das System zum Einbau unnatürlicher Aminosäuren durch Amber Stopp Kodons zur Verfügung stand. Im Anschluss wurden die Mutanten in der TM6 erzeugt und ihr Expression gezeigt. Abschließend wurden die Grundzüge einer Reinigung des modifizierten Proteins etabliert.
Zusammenfassend hat diese Arbeit die Grundlagen für die gezielte Untersuchung der Substratbindungsstelle des ABC-Transporters LmrA aus L. lactis geschaffen und stellt die Basis für eine Vielzahl von Experimenten zum Verständnis des Transportprozesses von LmrA dar.
Referenzen
Alqawi, O., G. Poelarends, et al. (2003). "Photoaffinity labeling under non-energized conditions of a specific drug-binding site of the ABC multidrug transporter LmrA from Lactococcus lactis." Biochem Biophys Res Commun 311(3): 696-701.
Altenberg, G. A., C. G. Vanoye, et al. (1994). "Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane." Proc Natl Acad Sci U S A 91(11): 4654-7.
Arechaga, I., B. Miroux, et al. (2000). "Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase." FEBS Lett 482(3): 215-9.
Bergseid, M., B. Scott, et al. (1991). "A high fidelity thermostable DNA polymerase isolated from Pyrococcus jiiriosus." Strategies in Molecular Biology 4: 34-35.
Birnboim, H. C. und J. Doly (1979). "A rapid alkaline extraction procedure for screening recombinant plasmid DNA." Nucleic Acids Res 7(6): 1513-23.
Blake, M. S., K. H. Johnston, et al. (1984). "A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots." Anal Biochem 136(1): 175-9.
Bradford, M. M. (1976). "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding." Anal Biochem 72: 248-54.
Chang, G., C. B. Roth, et al. (2006). "Retraction." Science 314(5807): 1875.
Chung, C. T., S. L. Niemela, et al. (1989). "One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution." Proc Natl Acad Sci U S A 86(7): 2172-5.
Dawson, R. J. und K. P. Locher (2006). "Structure of a bacterial multidrug ABC transporter." Nature 443(7108): 180-5.
Federici, L., B. Woebking, et al. (2007). "New structure model for the ATP-binding cassette multidrug transporter LmrA." Biochem Pharmacol 74(5): 672-678.
Gasteiger, E., A. Gattiker, et al. (2003). "ExPASy: The proteomics server for in-depth protein knowledge and analysis." Nucleic Acids Res 31(13): 3784-8.
Hanahan, D. (1983). "Studies on transformation of Escherichia coli with plasmids." J Mol Biol 166(4): 557-80.
Hanekop, N. (2006). Strukturbiologische Charakterisierung des ABC-Transporters LmrA aus L. lactis und des Substratbindeproteins EhuB aus S. meliloti. Institut für Biochemie. Frankfurt, Johann Wolfgang Goethe Universität Frankfurt.
Hanrahan, J. W., M. Gentzsch, et al. (2002). "The cystic fibrosis transmembrane conductance regulator (ABCC7)." ABC Proteins: From Bacteria to Man: 589–618.
Higgins, C. F. und M. M. Gottesman (1992). "Is the multidrug transporter a flippase?" Trends Biochem Sci 17(1): 18-21.
Higgins, C. F. u. L., K.J (2003). ABC transporters: an introduction and overview. ABC proteins: from bacteria to man. I. B. Holland, Cole, S.P., Kuchler, K. und Higgins, C.F. (eds.), Academic Press, Amsterdam.
Juliano, R. L. und V. Ling (1976). "A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants." Biochim Biophys Acta 455(1): 152-62.
Laemmli, U. K. (1970). "Cleavage of structural proteins during the assembly of the head of bacteriophage T4." Nature 227(5259): 680-5.
Lovell, S. C., I. W. Davis, et al. (2003). "Structure validation by Calpha geometry: phi,psi and Cbeta deviation." Proteins 50(3): 437-50.
Margolles, A., M. Putman, et al. (1999). "The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids." Biochemistry 38(49): 16298-306.
Matsuo, M., K. Ueda, et al. (2003). "The sulfonylurea receptor: an ABCC transporter that acts as an ion channel regulator." ABC Proteins from Bacteria to Man: 551–575.
McClelland, M. und M. Nelson (1992). "Effect of site-specific methylation on DNA modification methyltransferases and restriction endonucleases." Nucleic Acids Res 20 Suppl: 2145-57.
Miroux, B. und J. E. Walker (1996). "Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels." J Mol Biol 260(3): 289-98.
Notredame, C., D. G. Higgins, et al. (2000). "T-Coffee: A novel method for fast and accurate multiple sequence alignment." J Mol Biol 302(1): 205-17.
Poelarends, G. J. und W. N. Konings (2002). "The transmembrane domains of the ABC multidrug transporter LmrA form a cytoplasmic exposed, aqueous chamber within the membrane." J Biol Chem 277(45): 42891-8.
Poirot, O., E. O'Toole, et al. (2003). "Tcoffee@igs: A web server for computing, evaluating and combining multiple sequence alignments." Nucleic Acids Res 31(13): 3503-6.
Raviv, Y., H. B. Pollard, et al. (1990). "Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells." J Biol Chem 265(7): 3975-80.
Ryu, Y. und P. G. Schultz (2006). "Efficient incorporation of unnatural amino acids into proteins in Escherichia coli." Nat Methods 3(4): 263-5.
Saiki, R. K., D. H. Gelfand, et al. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science 239(4839): 487-91.
Sali, A. und T. L. Blundell (1993). "Comparative protein modelling by satisfaction of spatial restraints." J Mol Biol 234(3): 779-815.
Sanger, F., S. Nicklen, et al. (1977). "DNA sequencing with chain-terminating inhibitors." Proc Natl Acad Sci U S A 74(12): 5463-7.
Saurin, W., M. Hofnung, et al. (1999). "Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters." J Mol Evol 48(1): 22-41.
Stryer, L. (2002). Biochemistry. Fifth, New York: Freeman [&] Co.
Studier, F. W. und B. A. Moffatt (1986). "Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes." J Mol Biol 189(1): 113-30.
van Veen, H. W., R. Callaghan, et al. (1998). "A bacterial antibiotic-resistance gene that complements the human multidrug-resistance P-glycoprotein gene." Nature 391(6664): 291-5.
van Veen, H. W., K. Venema, et al. (1996). "Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1." Proc Natl Acad Sci U S A 93(20): 10668-72.
Wang, J., J. Xie, et al. (2006). "A genetically encoded fluorescent amino acid." J Am Chem Soc 128(27): 8738-9.
Wang, L., A. Brock, et al. (2001). "Expanding the genetic code of Escherichia coli." Science 292(5516): 498-500.
Xie, J. und P. G. Schultz (2006). "A chemical toolkit for proteins--an expanded genetic code." Nat Rev Mol Cell Biol 7(10): 775-82.