Research Research Training Training

Yu Xiao DiYu Xiao DiUppsala UniversityUppsala University

ChaperoneChaperone

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

Chaperone - subunit Chaperone - subunit complex complex

Subunit – subunit Subunit – subunit complexcomplex

How do they work ?How do they work ?

Our taskOur task

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

Research workResearch work

Protein expression

Purification

Crystallization of Caf1M-Caf1’-Caf1’’ complex

X-ray diffraction

Protein expressionProtein expression

Host strain Host strain E. coliE. coli DH5 DH5αα strain was t strain was transformed with pFM1-1-T7F. For expreransformed with pFM1-1-T7F. For expression of mutated complexes, cells werssion of mutated complexes, cells were grown to an OD600nm of 0.8-1 in LB e grown to an OD600nm of 0.8-1 in LB medium ( Maniatismedium ( Maniatis et al et al., 1998) conta., 1998) containing 60 ining 60 mgmg L–1 ampicillin. Protein e L–1 ampicillin. Protein expression was induced with 0.3-0.4 mxpression was induced with 0.3-0.4 mMM IPTG for 2 h.IPTG for 2 h.

Purification complexPurification complex Samples were loaded onto an 8 Samples were loaded onto an 8 ml ml Mono Q Mono Q column. Fractions were analyzed by isoecolumn. Fractions were analyzed by isoelectric focusing ( IEF) on pH 4-6 gradilectric focusing ( IEF) on pH 4-6 gradient gels to detect the target complex. ent gels to detect the target complex. Samples were concentrated and incubated Samples were concentrated and incubated with TAGZym at 33°overnight. Digestion ewith TAGZym at 33°overnight. Digestion efficiency was checked by IEF. Samples wfficiency was checked by IEF. Samples were dialysis against 40 mere dialysis against 40 mMM NaAC, pH 4. NaAC, pH 4.6-4.7 and loaded into a 1 6-4.7 and loaded into a 1 mlml Mono S equ Mono S equilibrated column with 40 milibrated column with 40 mMM NaAC, pH 4. NaAC, pH 4.6-4.7 . Sample was concentrated to 30 6-4.7 . Sample was concentrated to 30 mmg/ml g/ml on a 2 on a 2 mlml VivaSpin device with MW VivaSpin device with MW cut off of 10 cut off of 10 kDakDa, 2050 , 2050 rpm.rpm.

Purification curve Purification curve

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

Photograph of the Photograph of the crystalcrystal

Photograph of the crystal Photograph of the crystal

Photograph of the Photograph of the crystalcrystal

X-ray diffraction studyX-ray diffraction study Diffraction data were collected under liquid-nitroDiffraction data were collected under liquid-nitro

gen cryoconditions at 100 K. To avoid damage on frgen cryoconditions at 100 K. To avoid damage on freezing, crystals were soaked for 10-30 s in a cryoeezing, crystals were soaked for 10-30 s in a cryoprotection solution containing 14 protection solution containing 14 %% PEG 400, 11 PEG 400, 11 %% PEG 8000, 60 PEG 8000, 60 mMmM NaAC, 120 NaAC, 120 mMmM Ca(AC)2, pH 5.7-6.0. Ca(AC)2, pH 5.7-6.0. Crystals were flash-cooled by rapidly moving them Crystals were flash-cooled by rapidly moving them into the cold nitrogen stream or by dipping them iinto the cold nitrogen stream or by dipping them in liquid nitrogen. X-ray diffraction data were coln liquid nitrogen. X-ray diffraction data were collected using rotating anode as an X-ray source (lected using rotating anode as an X-ray source (λλ==1.45 1.45 ÅÅ) and recorded on a Mar-345 detector. Images ) and recorded on a Mar-345 detector. Images of the diffraction of the crystals were analyzed tof the diffraction of the crystals were analyzed to find preliminary space group to be either P222 oo find preliminary space group to be either P222 or P2r P21122112211 or P2 or P21122112 or P2222 or P22211 with cell unit a=71.2 with cell unit a=71.2 ÅÅ,, b=96.5 b=96.5 ÅÅ, c=166.4 , c=166.4 ÅÅ, , αα==ββ==γγ =90°. It was predicte =90°. It was predicted that asymmetric unit contains two molecules.d that asymmetric unit contains two molecules.

The image of the The image of the diffraction diffraction

The image of the The image of the diffractiondiffraction

Wake up ! Story is not Wake up ! Story is not endingending

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

AcknowledgmentsAcknowledgments

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

ReferenceReference Anton V. Zavialov, Jenny Berglund (2003). Structure and biAnton V. Zavialov, Jenny Berglund (2003). Structure and bi

ogenesis of the capsular F1 Antigen from ogenesis of the capsular F1 Antigen from Yersinia pestisYersinia pestis: : Preserved Folding Energy Drives Fiber Formation. Preserved Folding Energy Drives Fiber Formation. CellCell 113: 113:587-596.587-596.

A. V. Zavialov, J. Kersley (2002). Donor strand complementA. V. Zavialov, J. Kersley (2002). Donor strand complementation mechanism in the biogenesis of non-pilus systems. ation mechanism in the biogenesis of non-pilus systems. MoMolecular microbiologylecular microbiology 45(4): 983-995. 45(4): 983-995.

Frederic G. Sauer, Stefan D. Knight (2000). PapD-like chapFrederic G. Sauer, Stefan D. Knight (2000). PapD-like chaperones and pilus biogenesis. erones and pilus biogenesis. Cell & Developmental biologyCell & Developmental biology 2000:pp.27-34.2000:pp.27-34.

Anton V. Zavialov, Vladimir M. Tischenko (2005). Resolving Anton V. Zavialov, Vladimir M. Tischenko (2005). Resolving the energy paradox of chaperone/usher-mediated fibre assemthe energy paradox of chaperone/usher-mediated fibre assembly.bly.Biochem.JBiochem.J.389:685-694..389:685-694.

Devapriya Choudhury, Andrew Thompson (1999). X-ray StructuDevapriya Choudhury, Andrew Thompson (1999). X-ray Structure of the FimC-FimH Chaperone-Adhesin Complex from Uropathre of the FimC-FimH Chaperone-Adhesin Complex from Uropathogenic Escherichia coli. ogenic Escherichia coli. ScienceScience 285:1061-1066. 285:1061-1066.

Stefan D Knight, Jenny Berglund (2000). Bacterial adhesins:Stefan D Knight, Jenny Berglund (2000). Bacterial adhesins: structureal studies reveal chaperone function and pilus b structureal studies reveal chaperone function and pilus biogenesis. iogenesis. Chemical Biology Chemical Biology 4:653-660.4:653-660.

Frederic G Sauer, Michelle Barnhart (2000). Chaperone-assiFrederic G Sauer, Michelle Barnhart (2000). Chaperone-assisted pilus assembly and bacterial attachment. sted pilus assembly and bacterial attachment. Structural bStructural biology iology 10:548-556.10:548-556.

Good Luck in the exam Good Luck in the exam coming next week !!!coming next week !!!

Thank you.Thank you. 12-17-200512-17-2005