Research Training

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Research Training

• Yu Xiao Di
• Uppsala University

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Chaperone

• A protein that helps other proteins to
  fold.Molecular chaperones do not specify the
  tertiary structure of a protein, they merely help
  the protein find that correct structure. They
  prevent protein aggregation by holding the
  protein in an open conformation until it is
  completely synthesized and ready to fold.

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Chaperone - subunit complex
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Subunit subunit complex
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How do they work ?
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Our task

• Our task is to understand the structure of
  virulence organelles. We decided to determine a
  minimal fiber of F1 capsular antigen. This fiber
  is a complex containing one molecule of the Caf1M
  chaperone and two molecules of Caf1 subunit
  (Caf1M-Caf1-Caf1).

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Research work
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Protein expression

• Host strain E. coli DH5a strain was transformed
  with pFM1-1-T7F. For expression of mutated
  complexes, cells were grown to an OD600nm of
  0.8-1 in LB medium ( Maniatis et al., 1998)
  containing 60 mg L1 ampicillin. Protein
  expression was induced with 0.3-0.4 mM IPTG for 2
  h.

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Purification complex

• Samples were loaded onto an 8 ml Mono Q column.
  Fractions were analyzed by isoelectric focusing (
  IEF) on pH 4-6 gradient gels to detect the target
  complex. Samples were concentrated and incubated
  with TAGZym at 33overnight. Digestion efficiency
  was checked by IEF. Samples were dialysis against
  40 mM NaAC, pH 4.6-4.7 and loaded into a 1 ml
  Mono S equilibrated column with 40 mM NaAC, pH
  4.6-4.7 . Sample was concentrated to 30 mg/ml on
  a 2 ml VivaSpin device with MW cut off of 10 kDa,
  2050 rpm.

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Purification curve
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Crystallization

• Crystallization was performed by the hanging-drop
  vapour-diffusion method at 293 K. In all
  experiments the crystallization drops, containing
  1.5 µl protein solution (diluted) and 1.5 µl
  precipitant solution, were equilibrated against 1
  ml precipitant solution. The initial
  crystallization conditions were found using
  Hampton Crystal Screen. Small crystals were found
  in condition 46. Best crystals were found in
  droplets containing 10 PEG 8000, 0.5 M NaAC,
  0.1 M Ca(AC)2, pH 5.7-6.0.

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Photograph of the crystal
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Photograph of the crystal
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Photograph of the crystal
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X-ray diffraction study

• Diffraction data were collected under
  liquid-nitrogen cryoconditions at 100 K. To avoid
  damage on freezing, crystals were soaked for
  10-30 s in a cryoprotection solution containing
  14 PEG 400, 11 PEG 8000, 60 mM NaAC, 120 mM
  Ca(AC)2, pH 5.7-6.0. Crystals were flash-cooled
  by rapidly moving them into the cold nitrogen
  stream or by dipping them in liquid nitrogen.
  X-ray diffraction data were collected using
  rotating anode as an X-ray source (?1.45 Å) and
  recorded on a Mar-345 detector. Images of the
  diffraction of the crystals were analyzed to find
  preliminary space group to be either P222 or
  P212121 or P21212 or P2221 with cell unit a71.2
  Å, b96.5 Å, c166.4 Å, aß? 90. It was
  predicted that asymmetric unit contains two
  molecules.

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The image of the diffraction
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The image of the diffraction
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Wake up ! Story is not ending

• Complete diffraction data set using one of the
  frozen crystals will be collected at synchrotron
  in France. And then the structure of the protein
  complex will be solved by molecular replacement
  (MOLREP). Later, other characters of this complex
  and another mutated protein complexes will be
  studied.

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Acknowledgments

• I would like to thank Stefan Knight for letting
  me do my research training in his group. I also
  want to thank my supervisor Anton Zavialov for
  helping me solve lots of problems.

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Reference

• Anton V. Zavialov, Jenny Berglund (2003).
  Structure and biogenesis of the capsular F1
  Antigen from Yersinia pestis Preserved Folding
  Energy Drives Fiber Formation. Cell 113587-596.
• A. V. Zavialov, J. Kersley (2002). Donor strand
  complementation mechanism in the biogenesis of
  non-pilus systems. Molecular microbiology 45(4)
  983-995.
• Frederic G. Sauer, Stefan D. Knight (2000).
  PapD-like chaperones and pilus biogenesis. Cell
  Developmental biology 2000pp.27-34.
• Anton V. Zavialov, Vladimir M. Tischenko (2005).
  Resolving the energy paradox of
  chaperone/usher-mediated fibre assembly.Biochem.J.
  389685-694.
• Devapriya Choudhury, Andrew Thompson (1999).
  X-ray Structure of the FimC-FimH
  Chaperone-Adhesin Complex from Uropathogenic
  Escherichia coli. Science 2851061-1066.
• Stefan D Knight, Jenny Berglund (2000). Bacterial
  adhesins structureal studies reveal chaperone
  function and pilus biogenesis. Chemical Biology
  4653-660.
• Frederic G Sauer, Michelle Barnhart (2000).
  Chaperone-assisted pilus assembly and bacterial
  attachment. Structural biology 10548-556.

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• Good Luck in the exam coming next week !!!
• Thank you.
• 12-17-2005