Research Training - PowerPoint PPT Presentation

Loading...

PPT – Research Training PowerPoint presentation | free to download - id: c37cf-ZDc1Z



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Research Training

Description:

Photograph of the crystal. Photograph of the crystal ... X-ray Structure of the FimC-FimH Chaperone-Adhesin Complex from Uropathogenic Escherichia coli. ... – PowerPoint PPT presentation

Number of Views:15
Avg rating:3.0/5.0
Slides: 22
Provided by: Dee148
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Research Training


1
Research Training
  • Yu Xiao Di
  • Uppsala University

2
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.

3
Chaperone - subunit complex
4
Subunit subunit complex
5
How do they work ?
6
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).

7
Research work
8
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.

9
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.

10
Purification curve
11
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.

12
Photograph of the crystal
13
Photograph of the crystal
14
Photograph of the crystal
15
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.

16
The image of the diffraction
17
The image of the diffraction
18
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.

19
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.

20
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.

21
  • Good Luck in the exam coming next week !!!
  • Thank you.
  • 12-17-2005
About PowerShow.com