Summer Research Crystallization of the Lysozyme Crystallization of the Lysozyme The Structure of HDV...

Summer ResearchSummer Research

•• Crystallization of the LysozymeCrystallization of the Lysozyme

•• The Structure of HDV AntigenThe Structure of HDV Antigen

Institute of Molecular Biology Academic Sinica R.O.C

Dr. Chwan-Deng (David) Hsiao

Winnie Charng (2002/7~8)

Dr. Chwan-Deng (David) Hsiao Dr. Chwan-Deng (David) Hsiao

Crystallographic Studies of Various Biological Macromolecules

1982 B.S. Dept. Chemistry, Chung-Yuan Christian Univ. 1984 M.S. Dept. Chemistry, Natl. Taiwan univ. 1993 Ph.D. Dept. Crystallography, Univ.of Pittsburgh, USA 1993-95 PDF IMB, Academia Sinica2/95-5/99 　 Assistant Research Fellow, IMB5/99-present 　 Associate Research Fellow, IMB

Recent Research: Hsc 70, HDV antigen , Phosphoglucose isomerase and Toc 34

Hsc70 is a chaperonin in cytosol and is composed of three domains. Currently, the C-terminal 10-KDa fragment is crystallized. However, its functional role needs to be determined. With this structure, the lab would like to explain experimental data and elucidate a protein-protein interaction of hsc-70 system.

Phosphoglucose isomerase (PGI) is a bifunctional enzyme playing a central role in glycolysis and gluconeogenesis . More interesting, the PGI isolated from pig muscle shares 90% homologous in amino acid sequences to mouse neuroleukin, a neurotrophic factor supporting the survival of the neurons. Recent data shows that murine autocrine motility factor exhibits the enzymatic properties of PGI, and 6-phosphogluconate inhibited both enzymatic activity and AMF-induced cell motility. The lab expects to answer the key questions about the structure-function of PGI and its relationship to Neuroleukin and Tumor cell Autocrine Motility Factor.

Toc 34 is a member of the outer membrane translocon complex that mediates the initial stage of protein import into chloroplasts.Toc34 is proposed to regulate the gating properties of Toc75 or regulate the recognition and presents precursor proteins. Besides, Toc34 is a GTP-binding protein. It is anchored in the outer membrane through the C-terminal hydrophobic domain and the GTP-binding domain is exposed in the cytosol. The structure information of the Toc components will provide better understanding of how the Toc complex functions to mediate precursor protein import. .

•• Crystallization of the LysozymeCrystallization of the Lysozyme＊＊ Introduction Introduction ＊＊ PrinciplePrinciple＊＊ Methods and MaterialsMethods and Materials＊＊ ResultResult

•• The Structure of HDV AntigenThe Structure of HDV Antigen＊＊ IntroductionIntroduction＊＊ PrinciplePrinciple＊＊ Materials and MethodsMaterials and Methods＊＊ Result Result ＊＊ DiscussionDiscussion＊＊ Future WorkFuture Work

＊ IntroductionLysozyme ：　 It hydrolyzes the β-

1,4 glucosidic linkages between NAM and NAG in the cell wall of certain microorganisms.

Crystallization of the LysozymeCrystallization of the Lysozyme

＊ Principle Crystallization ： formation of solid crystals from a homogeneous solution.Process ： (1) nucleation--the growth of a new crystal. To initiate the process, supersaturation driving force is necessary. (2) crystals grow gradually by surface interaction with the solute.


A common approach is vapor diffusion ：　　　　　　 (1)sitting drop (2)hanging drop

[ppt]drop= [ppt]reservoir/2

[ppt]drop= [ppt]reservoir

HANGING DROP

SITTING DROP


＊ Material and Method Solution A: 6% lys(0.1M CH3COONa) Solution B: 15% NaCl(0.1M CH3COONa) Sitting Drop: Every well 5λA+5λB,

reservoir B 1ml, 25℃ Hanging Drop ： Every well 1λA+1λB (in cover slips), 500λB, 25℃


＊ Results

The next day, I found that in all wells there were lots of large clear crystals with cubic, tetragonal, hexagonal, and trigonal shapes. In addition, the crystals in the sitting drop are bigger than that in the hanging drop.



Hexagonal

•• Crystallization of the LysozymeCrystallization of the Lysozyme＊＊ Introduction Introduction ＊＊ PrinciplePrinciple＊＊ Methods and MaterialsMethods and Materials＊＊ ResultResult

•• The Structure of HDV AntigenThe Structure of HDV Antigen＊＊ IntroductionIntroduction＊＊ PrinciplePrinciple＊＊ Materials and MethodsMaterials and Methods＊＊ Result Result ＊＊ DiscussionDiscussion＊＊ Future WorkFuture Work

＊ Introduction HDV (hepatitis D virus), a satellite virus of hepatitis B virus. The encoded delta antigen is a nuclear phosphoprotein with RNA binding activities in regions near N terminus (residue 24~50). The crystal structure from residue 12 to 60 has been solved, and now we try to find how this region binds with polynucleotides.

The Structure of HDV AntigenThe Structure of HDV Antigen

＊ Principle Ion-exchange chromatography: The charged resins interact differently with various proteins, thus separate them by charge. X-ray diffraction: The atomic planes of a crystal cause an incident beam of X-rays to interfere with one another as they leave the crystal and are detected and calculated to get the structure.

＊ Materials and Methods Buffer I:50mM hepes, 20% glycerol, pH 7.8 Buffer II:50mM hepes, 2M NaCl, 20% glycerol, pH 7.8 PET plasmids with Lac operon and AmpR Insert DNA corresponds to residue 14 to 59

of HDAg DNA segments from 9 to 23 nucleotides E.coli



TransformationTransformation(1) 2μl DNA+100μl competent cell (2) on ice 5 min(3) 42 2 min℃

(4) on ice 30 min

Cell cultureCell culture(1) +1ml LB(2) 37℃ shacking 2 hr(3) Transfer to 1L LB with 1ml amp(4) 37℃ shacking 12 hr


Get the proteinsGet the proteins(1) Collect cell pellet by centrifuge at 4℃, 4Krpm 20min

(2) Resuspend pellet in 15ml buffer I

(3) Microfuidizer to break the cells

(4) Centrifuge at 4℃, 25Krpm 40min

(5) Run PC column

(6) Wash with 0.5, 0.8M NaCl buffer(add buffer II to I)

(7) Elute with 1.2M NaCl buffer


ConcentrationConcentrationTransfer eluted solution to Amicon concentrator 5kd cutuntil the concentration larger than 10μg/μl (OD595nm

using BSA as standard protein)

DialysisDialysisJust dilute the salt NaCl

Set screenSet screen(1) Add protein solution to DNA fragment (1:1)

(2) Add 5M NaCl (1:2)


Modify conditionsModify conditions

X-ray diffractionX-ray diffraction

＊ Result I set screen I 、 II 、 III 、 V and found that microcrystals grew under about 11 conditions. (see the next page)

＊ Discussion As what we can see that there are no obvious common features between these conditions. Maybe the most important is that the protein has the supersaturated condition which drives the protein to aggregate orderly to become crystals.


4.6Na　Acetate

0.4M　(NH4)2HPO4 Needle

6.5　Na　Citrate 30%　PEG4K 0.2M　NH4OAc Rectangle

8.5　Tris 50%　MPD 0.2M　(NH4)2PO4 Aggregated　needle

5.5　Mes 5%　PEG8K 200mM　KCl 10mM　MgCl2 Rectangle

7.5　Tris 10%　MPD 10mM　MgCl2 Needle

7　Hepes 5%　PEG4K 200mM　NH4OAc 150mM　Mg(OAc)2 Needle

7　Hepes 10%　PEG400 100mM　KCl 10mM　CaCl2 Column

7　Hepes 10%　PEG400 100mMKCl 10mM　MgCl2 Needle

6.5　Cacodylate 10%　Hexanediol 5mM　MgCl2 0.1mM　Co(NH3)6Cl3 Needle

5.5　Mes 10%　PEG400 100mM　MgSO4 200mM　KCl Needle

3%MPD 0.02M　CaCl2 Snow　dust


＊ Future Work Microcrystals are not big enough for x-ray diffraction. The conditions I got are still needed further modifying. Besides, the N-terminal delta antigen used in the experiments are linked to His tag. Maybe cleaving this fusion protein to get pure portion of the protein that we actually want to solve is another way the lab can try to have the crystals.

Summer Research Crystallization of the Lysozyme Crystallization of the Lysozyme The Structure of HDV...

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