⺮ⓑ㉁ᕤᏛⓗᡭἲࢆ㥑౑ࡋࡓࢩࣕ࣌ࣟࢽࣥ GroEL ࡢ

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⺮

⺮ⓑ㉁ᕤᏛⓗᡭἲࢆ㥑౑ࡋࡓࢩࣕ࣌ࣟࢽࣥ GroEL ࡢ

࢔࣑ࣟ࢖ࢻ⥺⥔จ㞟ᢚไᶵ⬟㛤Ⓨ࡟㛵ࡍࡿ◊✲

201 㸵ᖺ 1 ᭶

⚟஭ ┤ஓ

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␎ ␎ྕ

Aș42㸸Amyloid

ș peptide

AD㸸GroEL apical domain ADP㸸adenosine diphosphate ATP㸸Adenosine triphosphate AFM㸸Atomic force microscope Amp㸸ampicillin

ANS㸸1-anilinonaphtalene-8-sulfonate BSA㸸bovine serum albmin

CD㸸circular dichroism DNA㸸deoxyribonucleic acid DTT㸸dithiothreitol

EDTA㸸ethylenediaminetetraacetic acid

ESC7㸸concatenated form of GroES where individual subunits were linked by a tri-glycine linker

G192X㸸Gly192 to X substitution GroEL mutants Gdn-HCl㸸guanidine hydrochloride

HSP㸸heat shockprotein MDH㸸malate dehydrogenase PD㸸parkinson’s disease

QCM㸸Quartz Crystal Microbalance assay Rho㸸Rhodanese

SDS-PAGE㸸sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC㸸Size exclusion chromatography

TEM㸸transmission electron microscopy

ThioT㸸Thioflavin T

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┠ ┠ḟ

➨ 1 ❶ ᗎㄽ ………...1

1-1㸬ࡣࡌࡵ࡟………..…………1

1-2㸬ࢩࣕ࣌ࣟࢽࣥ GroEL

ࡢᵓ㐀࡜ᶵ⬟………..……….2

1-3㸬ࢱࣥࣃࢡ㉁ࡢᵓ㐀␗ᖖ࡟ࡼࡾ⏕ࡌࡿ࢔࣑ࣟ࢖ࢻ⥺⥔จ㞟………..………..11

1-4㸬ࣃ࣮࢟ࣥࢯࣥ⑓Ⓨ⑕ࡢཎᅉࢱࣥࣃࢡ㉁Ș-Synuclein ………...15

1-5㸬ኳ↛ኚᛶ⺮ⓑ㉁࡟ᑐࡍࡿࢩࣕ࣌ࣟࢽࣥࡢാࡁ………..………..20

1-6㸬ᮏ◊✲ࡢ┠ⓗ………24

➨ 2 ❶ GroEL Gly192 ኚ␗యࢆ⏝࠸ࡓࢩࣕ࣌ࣟࢽࣥࡢᶵ⬟ゎᯒ … ………..25

2-1㸬⥴ゝ………..…….….25

2-2㸬ᮦᩱ࡜᪉ἲ……….…….…..26

2-2-1㸬ࣉࣛࢫ࣑ࢻ༢㞳……….26

2-2-2㸬࢔࣮࢞ࣟࢫࢤࣝ㟁ẼὋື………….………….………..26

2-2-3㸬ᙧ㉁㌿᥮……….………27

2-2-4㸬DNA Sequence……….………27

2-2-5㸬 GroEL G192X

ࡢస〇……….….…28

2-2-6㸬ࢱࣥࣃࢡ㉁ᐃ㔞……….…28

2-2-7㸬኱㔞ᇵ㣴……….…28

2-2-8㸬 GroEL(㔝⏕ᆺ㸪ྛ✀ኚ␗య)ࡢ⢭〇……….………..……...29

2-2-9㸬 CD

ᐃ……….………..………33

2-2-10㸬 ANS

⺯ග ᐃ……….………..………..33

2-2-11㸬ࢤࣝࢁ㐣ࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡿศ㞳ศᯒ………..34

2-2-12㸬 ATPase

άᛶ ᐃ……….………..……..35

2-2-13㸬 MDH

ࣜࣇ࢛࣮ࣝࢹ࢕ࣥࢢάᛶ ᐃ…….………..…….36

2-2-14㸬Rhodanese refolding assay………….………..………..37

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2-2-15㸬ศᏊ㛫┦஫స⏝ᐃ㔞(QCM)……….………..……39

2-2-16㸬 QCM

࡟ࡼࡿ࢝࢖ࢿࢸ࢕ࢵࢡࣃ࣓࣮ࣛࢱ࣮⟬ฟ………40

2-3㸬⤖ᯝ………43

2-3-1㸬㔝⏕ᆺ GroEL

࡜ྛ✀

G192X

ኚ␗యࡢᵓ㐀࡟㛵ࡍࡿゎᯒ………..….43

2-3-2㸬 ATPase

άᛶ ᐃ……….49

2-3-3㸬MDH㸪Rhodanese

ࣜࣇ࢛࣮ࣝࢹ࢕ࣥࢢάᛶ ᐃ………50

2-3-4㸬GroEL G192X

ኚ␗య࡜

GroES

ࡢ┦஫స⏝ゎᯒ………..52

2-4㸬⪃ᐹ………60

➨ ➨ 3 ❶ GroEL Gly192 ኚ␗య࡟ࡼࡿ࢔࣑ࣟ࢖ࢻ⥺⥔จ㞟ࡢᢚไຠᯝ ………..64

3-1

㸬⥴ゝ

……….…………64

3-2㸬ᮦᩱ࡜᪉ἲ……….………65

3-2-1㸬Ș-Synuclein

ࡢᇵ㣴………65

3-2-2㸬Ș-Synuclein

ࡢ⢭〇………66

3-2-3㸬 GroES

ࡢᇵ㣴࡜⢭〇………69

3-2-4㸬 ᐃ࡟⏝࠸ࡓࢧࣥࣉࣝࡢㄪᩚ……….………73

3-2-5㸬 QCM

࡟ࡼࡿኳ↛ኚᛶ⺮ⓑ㉁(Ș-Syn㸪

Aș)࡜ GroEL

ࡢ┦஫స⏝ゎᯒ………73

3-2-6㸬 QCM

࡟ࡼࡿ࢝࢖ࢿࢸ࢕ࢵࢡࣃ࣓࣮ࣛࢱ࣮⟬ฟ………..74

3-2-7㸬࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ……….………75

3-2-8㸬 GroEL

Ꮡᅾୗ࡟࠾ࡅࡿȘ-Synࡢ⥺⥔ᙧᡂ………..76

3-2-9㸬␗࡞ࡿ᫬㛫࡟࠾࠸࡚ GroEL

ࢆῧຍࡋࡓα-Synࡢ⥺⥔ᙧᡂ………..76

3-2-10㸬ཎᏊ㛫ຊ㢧ᚤ㙾(AFM) ᐃ……….…….……….76

3-2-11㸬㏱㐣ᆺ㟁Ꮚ㢧ᚤ㙾(TEM) ᐃ………….………..77

3-3㸬⤖ᯝ……….78

3-3-1㸬GroEL

࡜ኳ↛ኚᛶࢱࣥࣃࢡ㉁ࡢ┦஫స⏝ゎᯒ………..78

3-3-2㸬 GroEL

Ꮡᅾୗ࡟࠾ࡅࡿα-Synࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ………..81

3-3-3㸬␗࡞ࡿ᫬㛫࡟࠾ࡅࡿ GroEL

ࢆకࡗࡓȘ-Synࡢ⥺⥔ᙧᡂ……….88

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3-4㸬⪃ᐹ……….94

➨ ➨ 4 ❶ ࣑ࢽࢩࣕ࣌ࣟࣥࠕ GroEL-AD ࠖࢆ⏝࠸ࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂᢚไຠᯝ …...97

4-1㸬⥴ゝ……….97

4-2㸬ᮦᩱ࡜᪉ἲ……….99

4-2-1㸬 GroEL-AD

ࡢస〇………..99

4-2-2㸬 GroEL-AD

ࡢᇵ㣴࡜⢭〇………..99

4-2-3㸬 ᐃ࡟⏝࠸ࡓࢧࣥࣉࣝࡢㄪᩚ……….102

4-2-4㸬 GroEL-AD

ࢆకࡗࡓኚᛶࢱࣥࣃࢡ㉁ࡢ⥺⥔ᙧᡂᐇ㦂……….102

4-2-5㸬

࢔࣑ࣟ࢖ࢻ⥺⥔ࡢほᐹ………..103

4-2-6㸬 GroEL-AD

࡜ኚᛶࢱࣥࣃࢡ㉁ࡢ┦஫స⏝ゎᯒ……….103

4-3㸬⤖ᯝ………...105

4-3-1㸬 GroEL-AD

࡟ࡼࡿᵝࠎ࡞ࢱࣥࣃࢡ㉁࡬ࡢ┦஫స⏝࡜⥺⥔໬ᢚไ………..105

4-3-2㸬 GroEL-AD

ࡢᏑᅾୗ࡟࠾࠸࡚ᙧᡂࡉࢀࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧែほᐹ…………..107

4-3-3㸬␗࡞ࡿ᫬㛫࡟࠾ࡅࡿ GroEL-AD

ࢆకࡗࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ࡬ࡢᙳ㡪……….111

4-3-4㸬 GroEL-AD

࡜ᶆⓗࢱࣥࣃࢡ㉁࡜ࡢ┦஫స⏝ゎᯒ………..113

4-4㸬⪃ᐹ………..119

➨ 5 ❶ ⥲ྜ⪃ᐹ ……….122

5-1㸬 GroEL

ࡢᶵ⬟࡟࠾ࡅࡿ

Hinge2

㒊఩ࡢ

Gly192

ṧᇶࡢᙺ๭………122

5-2㸬

Ș-Synࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ࡟ᑐࡍࡿ

Apical domain

ࡢᙺ๭………..127

5-3㸬

ࢱࣥࣃࢡ㉁จ㞟࡟ᑐࡍࡿ

Apical domain

ࡢᙺ๭………130

➨ 6 ❶ ⤖ㄒ ………133

ᘬ⏝ᩥ⊩ ……….135

ㅰ㎡ ………..147

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1 ➨

➨ 1 ❶ ᗎㄽ

1-1 䠊䛿䛨䜑䛻

䝍䞁䝟䜽㉁䛿䠈20 ✀㢮䛛䜙䛺䜛䜰䝭䝜㓟ṧᇶ䛜ᯞศ䛛䜜䛩䜛䛣䛸䛺䛟୍ิ䛻䛴䛺䛜䛳䛶ฟ᮶ 䛶䛔䜛䚹඲䛶䛾䝍䞁䝟䜽㉁䛿䠈DNA 䛾ᣢ䛴㑇ఏ᝟ሗ䜢䜒䛸䛻స䜙䜜䛶䛚䜚䠈㑇ఏᬯྕ䛜ᣦᐃ 䛩䜛䛾䛿䠈䜰䝭䝜㓟㓄ิ䛾᝟ሗ䛰䛡䛷䛒䜛䚹⏕≀䜢ᙧస䛳䛶䛔䜛

20

✀㢮䛾䜰䝭䝜㓟ṧᇶ䜢䛹䜜䛰䛡䠈䛭䛧䛶䛹䛾䜘䛖䛺㡰ᗎ䛷୪䜉䜜䜀䠈୍ಶ䛾䝍䞁䝟䜽㉁䛸䛧䛶ᶵ⬟䜢ᯝ䛯䛩䜒䛾䛜䛷䛝䜛䛛䜢䠈᝟ሗ䛸䛧䛶ಖᏑ䛧䛶䛔䜛䛾䛜

DNA

䛷䛒䜛䚹䛴䜎䜚䠈䝠䝖䝀䝜䝮䛾඲ሷᇶ㓄ิ䛜᫂䜙䛛䛻䛺䛳䛯௒ ᪥ 䠈䝍䞁䝟䜽㉁䜢⌮ ゎ䛩䜛䛣䛸䛾㔜せ ᛶ 䛿䛣䜜䜎䛷䛻䛺䛟㧗䜎䛳䛶䛔䜛䚹㑇 ఏ ᝟ ሗ 䛿䠈

DNA䊻RNA䊻䝍䞁䝟䜽㉁䛸䛔䛖䝉䞁䝖䝷䝹䝗䜾䝬䜢⤒䛶䝍䞁䝟䜽㉁䜢⏕⏘䛩䜛䛜䠈䝸䝪䝋䞊䝮䛷

ྜᡂ䛥䜜䛯᪂⏕䝫䝸䝨䝥䝏䝗䛿༢䛻䜰䝭䝜㓟䛜୍ิ䛻୪䜉䜙䜜䛯⣣≧䛷䛒䜚䠈䛭䛾䜎䜎䛷䛿ᶵ

⬟䜢ᣢ䛯䛺䛔䚹䝍䞁䝟䜽㉁䛿ᅛ᭷䛾❧యᵓ㐀䛻ᢡ䜚䛯䛯䜏(䝣䜷䞊䝹䝕䜱䞁䜾)䛜⾜䜟䜜䜛䛣䛸䛷ጞ䜑䛶ᶵ⬟䜢ᣢ䛴䝍䞁䝟䜽㉁䛸䛺䜛䚹䝍䞁䝟䜽㉁䛿䝣䜷䞊䝹䝕䜱䞁䜾䛩䜛䛣䛸䛷⊂⮬䛾❧యᵓ 㐀䜢ᙧᡂ䛧䠈㓝⣲཯ᛂ䠈ච␿཯ᛂ䠈䜶䝛䝹䜼䞊ኚ᥮䠈≀㉁㍺㏦䠈⣽⬊㐠ື䛺䛹ከᙬ䛺⏕࿨

ᶵᵓ䜢ᢸ䛖䚹

䛧䛛䛧䛺䛜䜙䠈䝍䞁䝟䜽㉁䛿ᖖ䛻⏕࿨䛻᭷฼䛻స⏝䛧䛶䛔䜛䜟䛡䛷䛿䛺䛔䚹䝍䞁䝟䜽㉁䛾␗

ᖖᵓ㐀䜔䜰䝭䝻䜲䝗䛸䜘䜀䜜䜛␗ᖖ䛺ỿ╔䛜䜰䝹䝒䝝䜲䝬䞊⑓䜢䛿䛨䜑䛸䛩䜛⚄⤒ኚᛶ⑌ᝈ䛾 ཎᅉ䛾୍䛴䛸䛺䜛䛣䛸䛜㏆ᖺ᫂䜙䛛䛻䛺䛳䛶䛝䛶䛔䜛䚹1957 ᖺ䛻

Anfinsen

䜙䛿䠈RNaseA 㓝⣲䛻ᒀ⣲䜢ῧຍ䛧䛩䜛䛣䛸䛷䛭䛾❧యᵓ㐀䜢᏶඲䛻ቯ䛧䛯ᚋ䠈㏱ᯒ䛧䛶ᒀ⣲䜢㝖ཤ䛩䜛䛸䠈

⮬Ⓨⓗ䛺ᕳ䛝ᡠ䛧䛜㉳䛣䜛䛣䛸䛻䜘䜚άᛶ䛜ᅇ᚟䛩䜛䛣䛸䜢♧䛧䛯䚹䛣䜜䛻䜘䜚䠈䝍䞁䝟䜽㉁䛾❧

యᵓ㐀䛿䜰䝭䝜㓟㓄ิ䛥䛘Ỵ䜎䜜䜀Ỵᐃ䛩䜛䚹䛴䜎䜚䠈䝍䞁䝟䜽㉁䛾䝣䜷䞊䝹䝕䜱䞁䜾䛿௚䛛䜙䛾䜶䝛䝹䜼䞊䜢ᚲせ䛸䛧䛺䛔⮬Ⓨⓗ䛺䝥䝻䝉䝇䛷䛒䜛䛸䛔䛖䛂Anfinsen䛃䛾䝗䜾䝬䜢ᥦၐ䛧䛯(1)䚹䛧䛛䛧䠈䛣䛣䛷୍䛴ぢⴠ䛸䛥䜜䛶䛔䛯䛣䛸䛿䠈䝣䜷䞊䝹䝕䜱䞁䜾䛜⾜䜟䜜䜛⎔ቃ䛾ၥ㢟䛷䛒䛳䛯䚹

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2

ヨ㦂⟶䛾୰䛷ẚ㍑ⓗ䝍䞁䝟䜽㉁⃰ᗘ䛾ప䛔≧ែ䛷ᐇ㦂䜢⾜䛳䛯䛸䛝䛻䛿䠈Anfinsen 䛾䝗䜾䝬䛜ᡂ❧䛩䜛䛜䠈⣽⬊ෆ㒊䛾䜘䛖䛻䝍䞁䝟䜽㉁⃰ᗘ䛜㠀ᖖ䛻㧗䛔⎔ቃ䛻䛚䛔䛶䠈䛭䜜䛿ᚲ䛪䛧䜒ᡂ❧䛧䛺䛔䛣䛸䛜㏆ᖺ᫂䜙䛛䛸䛺䛳䛶䛝䛶䛔䜛䚹䛭䛣䛷Ⓩሙ䛩䜛䛾䛜ศᏊ䝅䝱䝨䝻䞁䛸࿧䜀䜜䜛୍⩌䛾䝍䞁䝟䜽㉁㞟ᅋ䛷䛒䜛䚹

11-2 䠊䝅䝱䝨䝻䝙䞁 GroEL 䛾ᵓ㐀䛸ᶵ⬟

1970

ᖺ௦䛻⇕䝅䝵䝑䜽䜢୚䛘䜛䛸䛭䛾⏕⏘㔞䛜ቑ䛘䜛䝍䞁䝟䜽㉁䛜ᩘ✀㢮☜ㄆ䛥䜜䛯䚹䛣䜜䜙䛾䝍䞁䝟䜽㉁䛿⇕ 䝅䝵䝑䜽䝍䞁䝟䜽㉁(Heat Shock Protein䠖HSP)䛸࿨ ྡ 䛥䜜䛯(2)䚹䛣䛾

HSP

䛿኱⭠⳦䛛䜙ே㛫䜎䛷䛩䜉䛶䛾⏕≀䛜ᣢ䛳䛶䛔䜛䛣䛸䛜ศ䛛䛳䛶䛔䜛䚹HSP 䛾䜰䝭䝜㓄

ิ䜢ẚ㍑䛧䛶䜏䜛䛸䠈኱⭠⳦䛸䝠䝖䛷䛭䛾┦ྠᛶ䛿

40 %

⛬ᗘ䛷䛒䜚䠈䝠䝖䛾

HSP

䛸኱⭠⳦䛾

HSP

䛿䜘䛟㢮ఝ䛧䛶䛔䜛䚹䝇䝖䝺䝇䛻ᑐ䛧䛶⣽⬊䜔ಶయ䜢Ᏺ䜛

HSP

䛿⏕≀䛻䛸䛳䛶᰿ᮏⓗ䠈䛛䛴ᚲせ୙ྍḞ䛺ാ䛝䜢䛧䛶䛚䜚䠈䛭䛾㐍໬䛾㐣⛬䛷䜘䛟ಖᏑ䛥䜜䛶䛝䛯䛣䛸䛜᥎ 䛥䜜䜛䚹㛗䛔⣣≧䛾䝫䝸䝨䝥䝏䝗䛷ྜᡂ䛥䜜䛯䝍䞁䝟䜽㉁䛿䠈⮬ศ⮬㌟䛾ຊ䛰䛡䛷䛿䛝䛱䜣䛸ᢡ䜚䛯䛯䜎䜜䛶ṇ䛧䛔❧యᵓ㐀䜢ᙧᡂ䛩䜛䛣䛸䛜䛷䛝䛺䛔䚹䛥䜙䛻䝍䞁䝟䜽㉁䛿䠈䛭䜜䛜ാ䛟ሙᡤ(䝭䝖䝁䞁䝗䝸䜰䜔ⴥ⥳య䛺䛹䛾⣽⬊ෆᑠჾᐁ)䛻⛣ື䛧䛺䛔㝈䜚䠈䛝䛱䜣䛸ാ䛟䛣䛸䛜䛷䛝䛺䛔䚹䛣䛾⛣

ື䜒䝍䞁䝟䜽㉁༢⊂䛷䛿䛷䛝䛺䛔䚹䛭䛣䛷䠈HSP 䛿䝍䞁䝟䜽㉁䜢䛝䛱䜣䛸䝣䜷䞊䝹䝕䜱䞁䜾䛧䛶 ṇ䛧䛔❧యᵓ㐀䛻䛧䛯䜚䠈⛣ື䜢ᡭఏ䛳䛯䜚䠈ኚᛶ䛧䛯䝍䞁䝟䜽㉁䜢䜒䛸䛾ṇ䛧䛔❧యᵓ㐀䛻 ᡠ䛩ᙺ๭䜢䛧䛶䛔䜛䚹䛣䛾䜘䛖䛻䠈䛒䛯䛛䜒䝍䞁䝟䜽㉁䛾䝅䝱䝨䝻䞁䛷䛒䜛䛛䛾䜘䛖䛻ᶵ⬟䛧䛶䛔䜛䛣䛸䛜ศ䛛䛳䛶䛝䛯

HSP

䛸䛔䛖ศᏊ䛿䠈䛂ศᏊ䝅䝱䝨䝻䞁䛃䛸䜘䜀䜜䜛䜘䛖䛻䛺䛳䛯(3)䚹

ศᏊ䝅䝱䝨䝻䞁䛿䠈HSP 䜔䜸䝸䝂䝬䞊䝍䞁䝟䜽㉁䛾㞟ྜ䛻㛵୚䛩䜛ᅉᏊ䛸䛧䛶Ⓨぢ䛥䜜䠈䛭䛾ᙺ๭䛾ゎ᫂䛸䛸䜒䛻ศᏊ䝅䝱䝨䝻䞁䛾ᴫᛕ䛜☜❧䛧䛯䚹ศᏊ䝅䝱䝨䝻䞁䛾ᙜึ䛾ᐃ⩏䛿䠈䝍䞁䝟䜽㉁䛾䝣䜷䞊䝹䝕䜱䞁䜾䜢ຓ䛡䜛୍⩌䛾䝍䞁䝟䜽㉁䛷䛒䜛䚹䛧䛛䛧䠈⌧ᅾ䛷䛿䝣䜷䞊䝹䝕䜱䞁䜾䛻䛸䛹䜎䜙䛪䠈䛥䜎䛦䜎䛺⣽⬊ᶵ⬟䛾ไᚚ䛻ศᏊ䝅䝱䝨䝻䞁䛜㛵୚䛧䛶䛔䜛䛣䛸䛜䜟䛛䛳䛶䛝䛶䛚䜚䠈ศᏊ䝅䝱䝨䝻䞁䛾ᴫᛕ䛿ᣑᙇ䛧䛶䛔䜛䚹

(11)

3

ཎ᰾⏕≀䛸┿᰾⏕≀䛾㛫䛷㐍໬ⓗ䛻䜘䛟ಖᏑ䛥䜜䛶䛔䜛ศᏊ䝅䝱䝨䝻䞁䛻䛿䠈HSP60 (䝅䝱䝨䝻䝙䞁

GroEL)䠈HSP70 (DnaK)䠈HSP90 (HtpG)䠈small HSP (IbpA/B)

䛺䛹䛾୍⩌

䛜䛒䜛䚹

E

䠊coli 䛾ሙྜ䠈䝸䝪䝋䞊䝮ୖ䛷ఙ㛗䛧䛴䛴䛒䜛䝫䝸䝨䝥䝏䝗䛾ከ䛟䛿䠈䝖䝸䜺䞊ᅉᏊ

(trigger factor䠖TF)䛸⤖ྜ䛧䠈ྜᡂ䛜᏶஢䛩䜛䛸䝸䝪䝋䞊䝮䛛䜙㞳䜜䛶䛔䛟䚹䛭䛾ᚋ䛿䠈䛥䜙䛻

ู䛾ศᏊ䝅䝱䝨䝻䞁䛻ཷ䛡Ώ䛥䜜䜛ሙྜ䛸䠈ศᏊ䝅䝱䝨䝻䞁䛾᥼ຓ䛺䛧䛷⮬Ⓨⓗ䛻䝣䜷䞊䝹䝕䜱䞁䜾䛩䜛䜒䛾䛻ศ䛛䜜䜛䚹⭷䝍䞁䝟䜽㉁䜢㝖䛟⣙

2500

✀䛾ྍ⁐ᛶ䝍䞁䝟䜽㉁䛾⣙

10

䠂䛿䝅䝱䝨䝻䝙䞁

(GroEL

䠋

GroES)

䝅䝇䝔䝮䛻䠈

5

䡚

18

㻌䠂䛿

HSP70

䠄

DnaK/DnaJ/GrpE

䠅䝅䝇䝔䝮䛻ຓ䛡䜙䜜䛶䝣䜷䞊䝹䝕䜱䞁䜾䛩䜛䛸䛔䜟䜜䛶䛚䜚䠈ṧ䜚䛾

70

䠂䛟䜙䛔䛾䝫䝸䝨䝥䝏䝗䛿䠈䝅䝱䝨䝻䞁䛾ຓ䛡䜢ᚲせ䛸䛧䛺䛔䛷⮬Ⓨⓗ䛻䝣䜷䞊䝹䝕䜱䞁䜾䛩䜛䛸⪃䛘䜙䜜䛶䛔䜛(4)䚹䝅䝱䝨䝻䝙䞁䛿䠈䜋䜌䛩䜉䛶䛾⣽⬊䛷⏕Ꮡ䛩䜛䛯䜑䛻ᚲ㡲䛷䛒䜛䚹䝅䝱䝨䝻䝙䞁䛿୍ḟ㓄ิ䛾┦ྠ

ᛶ䛿ẚ㍑ⓗప䛔䛜䠈❧యᵓ㐀ⓗ䛻䜘䛟ఝ䛯

2

䛴䛾䜾䝹䞊䝥䛻ศ㢮䛥䜜䛶䛚䜚䠈䜾䝹䞊䝥䊠ᆺ 䝅䝱䝨䝻䞁䛸䛧䛶

E . coli

䛺䛹䛾┿ṇ⣽⳦䜔䝭䝖䝁䞁䝗䝸䜰䠈ⴥ⥳య䛺䛹䛻Ꮡᅾ䛩䜛 HSP60

(GroEL)(5)䠈䜾䝹䞊䝥䊡ᆺ 䝅䝱䝨䝻䝙䞁䛸䛧䛶ྂ ⣽ ⳦ 䜔┿ ᰾ ⣽ ⬊ 䛾䝃䜲䝖䝌䝹䛻Ꮡ ᅾ 䛩䜛 CCT

䛺䛹䛜 ▱ 䜙䜜䛶䛔䜛䚹䜾䝹䞊䝥䊠 ᆺ 䝅䝱䝨䝻䝙䞁䛿㏻ ᖖ 䝁䝣䜯䜽䝍䞊䛸䛧䛶

HSP10 (GroES)

䜢ᚲせ䛸䛩䜛䛜䠈䜾䝹䞊䝥䊡ᆺ䝅䝱䝨䝻䝙䞁䛿

HSP10

䛻┦ᙜ䛩䜛㒊ศ䜢⮬ศ⮬㌟

䛾䝃䝤䝴䝙䝑䝖䛻ෆⶶ䛧䛯ᵓ㐀䜢ᙧᡂ䛧䛶䛔䜛(6)䚹

GroE

䛾䝣䜷䞊䝹䝕䜱䞁䜾䛾䝯䜹䝙䝈䝮䛿䠈䝅䝱䝨䝻䞁䛾䜸䝸䝂䝬䞊ᵓ㐀䛸ぶᐦ䛻㛵㐃䛧䛶䛔䜛䚹GroEL䛿

57 kDa

䛾䝃䝤䝴䝙䝑䝖

7

䛴䛛䜙䛺䜛䝸䞁䜾䛜⫼୰ྜ䜟䛫䛻

2

䛴㔜䛺䛳䛯

14

㔞 యᵓ㐀(⥲ศᏊ㔞䡚800 kDa)䜢ᙧᡂ䛧䛶䛔䜛(7)䚹GroEL 䛿┤ᚄ

137ύ䠈㧗䛥 146

ύ䛾኱

䛝䛥䛾෇⟄≧䛷䛒䜛䚹GroEL 䛷ᅖ䜎䜜䛯䝸䞁䜾ෆ㒊䛻䛿䛭䜜䛮䜜┤ᚄ⣙

45ύ

䛾✵Ὕ䛜䛒䜚䠈

2

䛴䛾✵Ὕ䛿䛴䛺䛜䛳䛶䛔䛺䛔䚹GroEL 䛾䛭䜜䛮䜜䛾䝃䝤䝴䝙䝑䝖䛿

3

䛴䛾䝗䝯䜲䞁䛛䜙䛺䛳䛶䛔䜛(ᅗ

1-1)(8)䚹

(12)

4

ᅗ1-1.䝅䝱䝨䝻䝙䞁䛾䝃䝤䝴䝙䝑䝖䛻䛚䛡䜛ྛ䝗䝯䜲䞁(8)

䜾䝹䞊䝥䊡ᆺ䝅䝱䝨䝻䝙䞁䛻䛿䜰䝢䜹䝹䝗䝯䜲䞁䛾ඛ➃䛻䜾䝹䞊䝥䊠ᆺ䛻䛿Ꮡᅾ䛧䛺䛔䝁䝅䝱䝨䝻䝙䞁䛾ᙺ๭

䜢ᯝ䛯䛩Helical protrusion䛜Ꮡᅾ䛧䛶䛔䜛䚹

䜶䜽䜰䝖䝸䜰䝹䝗䝯䜲䞁䛿䝉䞁䝖䝷䝹䜻䝱䝡䝔䜱䛾ෆഃ䛻ྥ䛛䛳䛶┤㠃䛧䛶䛔䜛

ATP

ຍỈศゎ䛾ゐ፹㒊఩䜢ྵ䜏䠈GroEL 䛾ᅵྎ䛷䛒䜛䚹ෆഃ䛾䝸䞁䜾䛾᥋ゐ䛸እഃ䛾䝸䞁䜾䛾᥋ゐ䛾኱㒊ศ 䛿䜶䜽䜰䝖䝸䜰䝹䝗䝯䜲䞁䛜௰௓䛧䛶䛔䜛䛯䜑䛻䜶䜽䜰䝖䝸䜰䝹䝗䝯䜲䞁䛿

GroEL

䜸䝸䝂䝬䞊䛾㔜せ䛺ᶵ⬟䜢ᯝ䛯䛩(9)䡝䜰䝢䜹䝹䝗䝯䜲䞁䛿✵Ὕ䛾ධཱྀ䛻఩⨨䛧䠈ከ䛟䛾␯Ỉᛶ䜰䝭䝜㓟ṧᇶ䛷 ᵓ ᡂ 䛥䜜䜛ᇶ ㉁䝫䝸䝨䝥䝏䝗䛸䛾⤖ ྜ 㒊 ఩ 䜢ྵ 䜣䛷䛔䜛(ྠ ᫬ 䛻

GroES

䛾⤖ ྜ 㡿 ᇦ 䜒ྵ

䜐)(10)䚹䜲䞁䝍䞊䝯䝕䜱䜶䜲䝖䝗䝯䜲䞁䛿

2

䛴䛾

Hinge

㒊఩䛻䜘䛳䛶䜶䜽䜰䝖䝸䜰䝹䝗䝯䜲䞁䛸䜰䝢䜹䝹䝗䝯䜲䞁䜢㐃⤖䛧䛶䛔䜛䚹ᚑ䛳䛶䠈䛭䛾୺せ䛺ᶵ⬟䛿୧᪉䛾㡿ᇦ䛾㛫䛾䜰䝻䝇䝔䝸䝑䜽ಙྕ

䛾ఏ㐩䛷䛒䜛(11)䚹

䝁䝅䝱䝨䝻䞁䛷䛒䜛

GroES

䛿┤ᚄ

75ύ䠈㧗䛥 30ύ

䛾䝗䞊䝮≧ᵓ㐀䜢䛧䛯

7

㔞య䛷䛒䜛

(12)䚹GroES

䛿

10kDa

䛾䝃䝤䝴䝙䝑䝖

7

䛴䛛䜙䛺䜛ᖗᏊ䛾䜘䛖䛺ᵓ㐀䜢ᙧᡂ䛧䛶䛔䜛䚹䛭䜜䛿䜋䛸䜣䛹䛜䃑䝅䞊䝖䛷ᵓᡂ䛥䜜䛶䛔䜛䚹GroES 䛾

16-33

␒┠䛾䜰䝭䝜㓟ṧᇶ䛿䜽䝷䝀䛾ゐᡭ 䛾䜘䛖䛺ᙧ䜢䛸䜚䠈GroEL 䛸┦஫స⏝䛩䜛䝰䝞䜲䝹䝹䞊䝥䛸࿧䜀䜜䜛㒊఩䛻┦ᙜ䛩䜛䚹GroES 䛿 GroEL 䛾䜰䝢䜹䝹䝗䝯䜲䞁䛻⤖ྜ䛧䠈䛣䛾⤖ྜ䛻䛿䠈䛭䜜䛮䜜䛾

GroEL

䝸䞁䜾䛻Ꮡᅾ䛩䜛䝚䜽䝺䜸䝏䝗⤖ྜ㒊఩䛻

ATP

䛛

ADP

䛜⤖ྜ䛧䛶䛔䜛䛣䛸䛜ᚲせ䛷䛒䜛

(13)䚹⤖ྜ䛾㝿䠈

(13)

5 GroES

䛾䝰䝞䜲䝹䝹䞊䝥䛿䠈GroEL 䛾␯Ỉᛶ䝨䝥䝏䝗䛸⤖ྜ䛩䜛䛣䛸䛷ᅛᐃ໬䛥䜜䜛䚹䛣䛾䜘䛖䛻䛧䛶䠈䝚䜽䝺䜸䝏䝗⤖ྜ䛸

GroES䠋䝫䝸䝨䝥䝏䝗⤖ྜ㛫䛷ᙉ䛔⤖ྜ䜢☜❧䛩䜛䚹䜰䝢䜹䝹

䝗䝯䜲䞁䛿䡚90°䜎䛷እ䛻ྥ䛛䛳䛶ᅇ㌿䛩䜛䚹␯Ỉᛶ᥋ྜ㒊఩䛿䝃䝤䝴䝙䝑䝖᥋ྜ㒊ศ䛻ᇙ䜑䜙䜜䠈✵Ὕ䛾ෆ㠃䜢ぶỈᛶ䛻䛧䛶䠈⤖ྜ䛧䛯䝫䝸䝨䝥䝏䝗䛾ᨺฟ䜢ᘬ䛝㉳䛣䛩䚹䛣䜜䛻క䛔䠈䜻䝱䝡䝔䜱䛾┤ᚄ䛿

45

䡚

80

ύ䜎䛷ቑຍ䛩䜛䚹

ADP

Ꮡᅾୗ䛷ᙧᡂ䛧䛯

GroEL-GroES

」ྜయ䛾❧యᵓ㐀䛜Ⓨ⾲䛥䜜䛶䛛䜙䝅䝱䝨䝻䞁

◊✲䛿୍Ẽ䛻㐍ᒎ䛧䛯(14)䚹䛭䛾ᵓ㐀䛿䠈GroEL 䛾∦᪉䛾䝸䞁䜾䛻

GroES

䛜⵹䛾䜘䛖䛻⤖

ྜ䛧䠈ྠ䛨ഃ䛾

7

䛴䛾

GroEL

䝃䝤䝴䝙䝑䝖䛻

1

䛴䛪䛴

ADP

䛜⤖ྜ䛧䛶䛔䛯(ᅗ

1-2)䚹

ᅗ1-2䠊GroEL-GroES-䠄ADP䠅䉀䛾X⥺⤖ᬗᵓ㐀(14) A䠊GroEL-GroES 」ྜయ䛾ୖ㠃ᅗ

B䠊GroEL-GroES 」ྜయ䛾ഃ㠃ᅗ C䠊GroEL-GroES 」ྜయ䛾㟁Ꮚᐦᗘᅗ

䛣䜜䛻䜘䜚ୖୗ䛾䝸䞁䜾䛿㠀ᑐ㇟䛻䛺䜛(GroES䠈ADP䛾⤖ྜ䛧䛯䝸䞁䜾䜢

cis

䉄ring䠈཯ᑐഃ䛾䝸䞁䜾䜢

trans

䉄ring 䛸䜘䜆)䚹䛥䜙䛻䠈

cis

-ring 䛾

GroEL

䝃䝤䝴䝙䝑䝖䛜䝎䜲䝘䝭䝑䜽䛷䠈䛭䛧䛶䝅䝱䝨䝻䞁䛾ᶵ⬟䜢⪃䛘䜜䜀⌮䛻䛛䛺䛳䛯ᵓ㐀ኚ໬䜢䛧䛶䛔䛯䚹䜲䞁䝍䞊䝯䝕䜱䜶䜲䝖䝗䝯䜲䞁䛾

(14)

6

୧➃䛻Ꮡᅾ䛩䜛

2

䛴䛾

Hinge

㒊఩䜢୰ᚰ䛻䜰䝢䜹䝹䝗䝯䜲䞁䛿ྑ䛻

90°

䜂䛽䜚䛺䛜䜙ୖ᪉ 䛻

60°

❧䛱ୖ䛜䜛(Openᆺ)䚹䛭䜜䛿䛱䜗䛖䛹䠈䛚㎡൤䜢䛧䛶䛔䜛ே䛜ᶓ䜢ྥ䛝䛺䛜䜙㉳䛝ୖ䜛䜘䛖䛺ឤ䛨䛷䛒䜛䚹䛣䛾ᵓ㐀ኚ໬䛾⤖ᯝ䠈

cis

䉄ringෆ䛾✵Ὕ䛾య✚䛜⣙

2

ಸ䛻ᗈ䛜䜚䠈GroES 䛾⵹䛻䜘䛳䛶኱䛝䛺㛢㙐✵㛫䛜⏕䜎䜜䜛䚹䛥䜙䛻䠈GroEL 䛾䝫䝸䝨䝥䝏䝗⤖ྜ㒊఩䛾䜋䛸䜣䛹䛜

GroES

䛸䛾⤖ྜ䛻⏝䛔䜙䜜䜛䚹䛭䛾䛯䜑䠈GroES 䛾⤖ྜ䛻䜘䛳䛶䝫䝸䝨䝥䝏䝗䛿

GroEL

䛛䜙ᘬ䛝䛿䛜䛥䜜䛶䠈ᣑ኱䛧䛯✵Ὕ䛻ᨺฟ䛥䜜䜛䛣䛸䛻䛺䜛䚹䝫䝸䝨䝥䝏䝗䛿䛣䛾✵Ὕ䛻ධ䜚㎸

䜐䛣䛸䛷䠈จ㞟䛾༴㝤䛛䜙㏨䜜䛶ṇᖖ䛺䝣䜷䞊䝹䝕䜱䞁䜾䛷䛝䜛

(

✵Ὕ䛻䛿⣙

57kDa

䜎䛷䛾䝫䝸䝨䝥䝏䝗䛜ධ䜚㎸䜑䜛)(15)䚹GroEL 䛜䝎䜲䝘䝭䝑䜽䛺ᵓ㐀ኚ໬䜢ᘬ䛝㉳䛣䛩䛣䛸䛻䜘䜚䠈

cis䉄 ring

䛾✵ Ὕ ෆ㒊䛿䝫䝸䝨䝥䝏䝗䛜䝣䜷䞊䝹䝕䜱䞁䜾䛩䜛䛾䛻㒔 ྜ䛾䜘䛔⎔ ቃ䛻ኚ ໬䛩䜛䚹

GroES

䛜⤖ྜ䛧䛶䛔䛺䛔䛸䛝䛻✵Ὕෆ㒊䛻㟢ฟ䛧䛶䛔䛯␯Ỉᛶ䜰䝭䝜㓟ṧᇶ䛿䠈GroES 䛾

⤖ྜ䛻䜘䛳䛶᪂䛯䛻ᙧᡂ䛥䜜䜛

GroEL

䝃䝤䝴䝙䝑䝖㛫䛾᥋ゐ⏺㠃䛷స⏝䛩䜛䚹⤖ᯝ䛸䛧䛶✵

Ὕෆ㒊䛿ぶỈⓗ䛺ṧᇶ䛜୺䛻䛺䜛䚹␯Ỉᛶ䜰䝭䝜㓟ṧᇶ䛜⾲㠃䛻᭚䛥䜜䛯᪂⏕ཪ䛿ኚᛶ䝫䝸䝨䝥䝏䝗䛜Ᏻᚰ䛧䛶ධ䜛䛣䛸䛜䛷䛝䜛✵㛫䜢䛴䛟䜛(16)䚹

䝅䝱䝨䝻஧䞁䛾཯ᛂ䝃䜲䜽䝹䛿䠈✵䛾

GroEL

䛻ኚᛶ≧ែ䛾ᇶ㉁䝫䝸䝨䝥䝏䝗䛜⤖ྜ䛩䜛䛣䛸䛛䜙ጞ䜎䜛(ᅗ

1-3A)䚹⥆䛔䛶䠈7

䛴䛾

ATP

䛜⤖ྜ䛧䠈GroEL䛜

Open

ᆺ䛻ᵓ㐀ኚ໬䛩䜛䚹䛣䜜䛻

GroES

䛜⤖ྜ䛩䜛䛣䛸䛷䠈䝫䝸䝨䝥䝏䝗䛿

cis䉄 ring

䛾✵Ὕෆ䛻ⴠ䛸䛧㎸䜎䜜䠈㝸㞳䛧䛯

✵㛫䛾୰䛷䝣䜷䞊䝹䝕䜱䞁䜾䛜㛤ጞ䛩䜛(

cis- ATP

」ྜయ

)䚹䛣䛾 cis- ATP

」ྜయ䛿

ATP

䛜 ຍỈศゎ䛧䛯

ADP( cis- ADP

」ྜయ)䛻䛺䜛䜎䛷Ᏻᐃ䛸䛺䜚䠈䝫䝸䝨䝥䝏䝗䛻䛸䛳䛶䛿䝅䝱䝨䝻䝙䞁䛾Ᏻ඲䛺✵Ὕෆ䛻䛚䛡䜛୍ᐃ䛾䝣䜷䞊䝹䝕䜱䞁䜾᫬㛫䛜ಖド䛥䜜䜛䛣䛸䛻䛺䜛䚹

cis䉄 ring

ෆ䛾

ATP

䛜ຍỈศゎ䛩䜛᫬㛫(⣙

8

⛊)䛜䝅䝱䝨䝻஧䞁䛾཯ᛂ䝃䜲䜽䝹䛾䝍䜲䝬䞊䛸䛧䛶ാ䛟䛾䛷䛒䜛䚹䛧䛛䛧䠈䛣䛾

8

⛊䛾䝍䜲䝬䞊䛿䛥䜙䛻

3

⛊䛸

5

⛊䛾

2

ẁ㝵䛻ศ䛡䜙䜜䜛䛣䛸䛜䜟䛛䛳䛯(ᅗ

1-3B)(17)䚹GroES

䛜⤖ྜ䛧䛶䛛䜙䝫䝸䝨䝥䝏䝗䛜✵Ὕෆ䛻ⴠ䛸䛧㎸䜎䜜䜛䜎䛷䛻᭱ึ

䛾

3

⛊䜢せ䛧䠈䛒䛸䛾

5

⛊䛷䝣䜷䞊䝹䝕䜱䞁䜾䛜㐍⾜䛩䜛䚹GroES 䛾

GroEL-ATP-ᇶ㉁䝍䞁

䝟䜽㉁」ྜయ䜈䛾⤖ྜ䛿ᇶ㉁䝍䞁䝟䜽㉁䛜䜻䝱䝡䝔䜱ෆ䛻᏶඲䛻ⴠ䛧㎸䜎䜜䛶䛔䛺䛔

cis-

(15)

7 ATP

^䖩」ྜయ䜢⏕䜏ฟ䛩䚹ATP 䛜

ADP

䛻ຍỈศゎ䛥䜜䠈

cis- ADP

」ྜయ䛻䛺䜛䛸䠈

trans䉄 ring

ഃ䛻

ATP

䛜⤖ྜ䛩䜛䚹䛣䛾

ATP

⤖ྜ䛜ᘬ䛝㔠䛸䛺䛳䛶䠈

cis䉄 ring

ഃ䛾

GroES

䛜ゎ㞳䛧䠈䛭䜜䛸ྠ᫬䛻

cis

䉄ring ഃ䛾

ADP

䛸୰䛻㛢䛨㎸䜑䜙䜜䛶䛔䛯䝫䝸䝨䝥䝏䝗䜒୍⥴䛻ゎ㞳䛩䜛䚹ኚᛶ䝫䝸䝨䝥䝏䝗䛿䠈䝣䜷䞊䝹䝕䜱䞁䜾䛜⤊஢䛧䛶䛔䜛䛛䛹䛖䛛䛻㛵䜟䜙䛪୍ᐃ᫬㛫䛷ᨺฟ 䛥䜜䜛䛜䠈䝣䜷䞊䝹䝕䜱䞁䜾㏵୰䛾䜒䛾䛿෌ᗘ䝅䝱䝨䝻஧䞁䛻ᤕ⋓䛥䜜䠈」ᩘᅇ䛾䝃䜲䜽䝹䜢

⤒䛯ᚋ䛻䝣䜷䞊䝹䝕䜱䞁䜾䛜᏶஢䛩䜛䚹௒䠈

trans䉄 ring

ഃ䛻䛿

ATP

䛸䝫䝸䝨䝥䝏䝗䛜⤖ྜ䛧䛶䛚䜚䠈௒ᗘ䛿䛣䛱䜙䛜

cis䉄 ring

ഃ䛸䛺䛳䛶ാ䛟䛣䛸䛻䛺䜛䚹䛣䛖䛧䛶䝃䜲䜽䝹䛿୍ᕠ䛩䜛

(18)

䚹

ᅗ1-3䠊GroEL䛾䝅䝱䝨䝻䝙䞁䝃䜲䜽䝹䝰䝕䝹(17) A䠊ᚑ᮶䛾䝅䝱䝨䝻䝙䞁཯ᛂ䝃䜲䜽䝹 B䠊2ẁ㝵䛾䝅䝱䝨䝻䝙䞁཯ᛂ䝃䜲䜽䝹

A A

^㻌

B

^㻌

(16)

8

䝅䝱䝨䝻䝙䞁

GroEL

䛿䝫䝸䝨䝥䝏䝗䜢ෆ㒊䛻ྲྀ䜚㎸䜐䛯䜑䛻኱ᖜ䛺ᵓ㐀ኚ໬䜢ᘬ䛝㉳䛣䛧䠈䝉䞁䝖䝷䝹䜻䝱䝡䝔䜱䛸࿧䜀䜜䜛✵Ὕ䜢㐀䜚ฟ䛩䚹䛣䛾㝿ᨭⅬ䛸䛺䜛䛾䛜ྛ䝗䝯䜲䞁㛫䛻Ꮡᅾ䛩䜛

Hinge

䛸࿧䜀䜜䜛㒊఩䛷䛒䜛䚹䜰䝢䜹䝹䝗䝯䜲䞁䛸䜲䞁䝍䞊䝯䝕䜱䜶䜲䝖䝗䝯䜲䞁䛾㛫䛻Ꮡᅾ䛩䜛

Hinge䊡㒊఩䛻 192

␒┠䛾

Gly

ṧᇶ(Gly192)䛜䛒䜛䚹௨๓䛻ᮏ◊✲ᐊ䛷䛿䠈Gly192 ṧᇶ 䜢

Trp

䛻⨨᥮䛧䛯

G192W

ኚ␗య䜢స〇䛧䠈䛭䛾䝅䝱䝨䝻䝙䞁ᶵ⬟䛾ゎᯒ䜢⾜䛳䛯

(19)

䚹䛭䛾⤖ᯝ䠈GroEL G192W ኚ␗య䛿

GroEL WT

䛸ẚ㍑䛧䛶ኚᛶ᮲௳ୗ䛾

Rhodanese

䜔

MDH

➼䛾ᇶ㉁䝍䞁䝟䜽㉁䛻ᑐ䛩䜛䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ䛜ⴭ䛧䛟పୗ䛧䛶䛔䜛⤖ᯝ䛜ᚓ䜙䜜䛶䛔䜛䚹GroEL G192W ኚ␗య䛾ᶵ⬟ゎᯒ䛾⤖ᯝ䜘䜚䠈Gly ṧᇶ䜢᭱䜒኱䛝䛔䜰䝭䝜㓟ṧ ᇶ䛷䛒䜛

Trp

䛻⨨᥮䛧䛯䛣䛸䛷∦᪉䛾

cis

䉄ring 䛜ண䜑

Open

≧ែ䛻䛺䜚䠈ATP 䛾㠀Ꮡᅾୗ

䛻䛚䛔䛶䜒

GroES

䜢⤖ྜ䛩䜛䛣䛸䛜䛷䛝䜛䛣䛸䛜᫂䜙䛛䛸䛺䛳䛯䚹䛥䜙䛻䠈䛣䛾䛸䛝䠈཯ᑐഃ䛾

trans䉄 ring

䝸䞁䜾䛻䛿ᇶ㉁䛜⤖ྜ䛩䜛䚹䛭䛾ᚋ䠈ATP 䛸㐣๫㔞䛾

GroES

䜢ῧຍ䛩䜛䛸ᇶ㉁

䛜እ䜜䠈௦䜟䜚䛻

GroES

䛜⤖ྜ䛩䜛䚹୧᪉䛾䝸䞁䜾䛻

GroES

䛜⤖ྜ䛧䛯䝣䝑䝖䝪䞊䝹ᆺ」ྜ

య䛸䛺䜛䛣䛸䛷䠈䛭䛾ᚋ䝅䝱䝨䝻䝙䞁䝃䜲䜽䝹䛜೵Ṇ䛧䛯䛂Dead-End䛃䛺≧ែ䛸䛺䜛(ᅗ

1-4)䚹䛣

䜜䜘䜚䠈GroEL G192Wኚ␗య䛿୧᪉䛾䝸䞁䜾䛷ྠ᫬䛻

GroES

䜢⤖ྜ䛩䜛䛣䛸䛜ྍ⬟䛸䛺䛳䛯䛜䠈ᇶ㉁䜢䝉䞁䝖䝷䝹䜻䝱䝡䝔䜱ෆ䛻ⴠ䛸䛧㎸䜐䛣䛸䛜ฟ᮶䛪䠈䝸䝣䜷䞊䝹䝕䜱䞁䜾䛾௓ຓ䜢⾜

䜟䛺䛔䛣䛸䛜᫂䜙䛛䛸䛺䛳䛯䚹

ᅗ1-4䠊GroEL G192Wኚ␗య䛾䝅䝱䝨䝻䝙䞁䝃䜲䜽䝹䛾ᴫ␎ᅗ(19)

(17)

9 GroEL

䛻䛚䛡䜛

2

䛴䛾䝸䞁䜾䛻ᑐ䛧䛶ྠ᫬䛻

GroES

䛜⤖ྜ䛧䛯䝣䝑䝖䝪䞊䝹ᆺ」ྜయ䛿䠈㠀⏕⌮ⓗ䛺᮲௳ୗ䛷䛾䜏⏕䛨䜛䛸⪃䛘䜙䜜䛶䛝䛯䚹䛧䛛䛧䠈㏆ᖺ䛻䛺䜚䛣䛾」ྜయ䛻ᑐ䛩䜛◊

✲䛿ቑຍ䛧䛶䛝䛶䛔䜛(ᅗ

1-5)䚹㔝⏕ᆺ GroEL

䛾⏕⌮᮲௳ୗ䛻䛚䛔䛶䠈䝣䝑䝖䝪䞊䝹ᆺ」ྜ

య䛜ᐇ㝿䛻ᙧᡂ䛥䜜䛶䛔䜛䛛䛿୙᫂䛺Ⅼ䛜ከ䛛䛳䛯䛜䠈⏕⌮ⓗ䛺᮲௳ୗ䛻䛚䛔䛶⺯ගඹ㬆䜶䝛䝹䜼䞊⛣ື

(FRET)

ἲ䛺䛹䜢⏝䛔䜛䛣䛸䛷䝣䝑䝖䝪䞊䝹ᆺ䛸ᙎ୸ᆺ䛜ྠ⛬ᗘᏑᅾ䛧䛶䛚䜚䠈

ADP/ATP

ẚ䛜㧗䛔䛸䝣䝑䝖䝪䞊䝹ᆺ䛾ᙧᡂ䛜㜼ᐖ䛥䜜䜛䛣䛸䜒ぢฟ䛥䜜䛶䛔䜛(20)䚹䛥䜙䛻䠈

398

␒┠䛾

Asp

䜢

Ala

䛻⨨᥮䛧䛯ኚ␗య䛷䛒䜛

GroEL D398A

ኚ␗య(21)䜢⏝䛔䛯◊✲

䛻䛚䛔䛶䜒䠈GroEL䛾୧᪉䛾䝸䞁䜾䛻

GroES

䛜⤖ྜ䛧䛯䝣䝑䝖䝪䞊䝹ᆺ」ྜయ䛜ᙧᡂ䛥䜜䜛䝰䝕䝹䜒⪃᱌䛥䜜䛶䛔䜛(22)䚹䛣䛾

GroEL D398A

ኚ␗య䛻ᑐ䛧

52

␒┠䛾

Asp

䜢

Ala

䛻

⨨᥮䛧䛯䛣䛸䛷䠈኱ᖜ䛻

ATP

䛾ຍỈศゎ䛜ᢚไ䛥䜜䛯

GroEL D398A/D52A

ኚ␗య䛿୧ ᪉䛾䝸䞁䜾䛻

GroES

䛜⤖ྜ䛧䛶䛔䜛䝣䝑䝖䝪䞊䝹ᆺ」ྜయ䛾

X

⥺⤖ᬗᵓ㐀䛜ゎ᫂䛥䜜䛶䛔䜛

(23)䚹䝅䝱䝨䝻䝙䞁䝃䜲䜽䝹䛻䛚䛡䜛䝣䝑䝖䝪䞊䝹ᆺ」ྜయ䛾Ꮡᅾ䛜☜ᐇ䛺䜒䛾䛸䛺䛳䛶䛔䜛䚹

ᅗ1-5䠊GroEL 䝣䝑䝖䝪䞊䝹」ྜయ䛾ᵓ㐀䛸཯ᛂ䝃䜲䜽䝹 A䠊GroEL D398A/D52A䠖GroES䈻」ྜయ䛾㟁Ꮚᐦᗘᅗ(23) 㻌㻌㻌㻌㻌㻌㻌㻌㻌B䠈C䠊ATP/ADP ẚ⋡䛾㐪䛔䛻䛚䛡䜛GroEL䛾䝅䝱䝨䝻䝙䞁䝃䜲䜽䝹(20)

B䠊పẚ⋡䛾ATP/ADP 䛻䛚䛡䜛䝅䝱䝨䝻䝙䞁䝃䜲䜽䝹 C䠊㧗ẚ⋡䛾ATP/ADP 䛻䛚䛡䜛䝅䝱䝨䝻䝙䞁䝃䜲䜽䝹

(18)

10 GroEL

䛾䜰䝢䜹䝹䝗䝯䜲䞁䛻⤖ྜ䛧䛶䛔䜛ᇶ㉁䛿䠈GroES 䛜⵹䜢䛩䜛䛣䛸䛷䝉䞁䝖䝷䝹䜻䝱䝡䝔䜱ෆ㒊䛻ⴠ䛸䛧㎸䜎䜜䛶䝸䝣䜷䞊䝹䝕䜱䞁䜾䛜௓ຓ䛥䜜䜛䚹䛭䛾ᚋ䠈ATP 䛜ຍỈศゎ䛥䜜䛶

ADP

䛸䛺䜛䛣䛸䛷

GroES

䛜እ䜜ᇶ㉁䛾ᨺฟ䛜⏕䛨䜛䚹䛣䛾⌧ᅾ⪃䛘䜙䜜䛶䛔䜛୍㐃䛾䝅䝱䝨䝻䝙䞁䝃䜲䜽䝹䛸䛿␗䛺䜚䠈GroES 䛜⵹䜢䛧䛯≧ែ䛷䜒䜻䝱䝡䝔䜱ෆ㒊䛾ᇶ㉁䛜እ䛻ᨺฟ䛧䠈䛭䛾ᚋᇶ㉁⮬య䛜⮬Ⓨⓗ䛻䝸䝣䜷䞊䝹䝕䜱䞁䜾䛩䜛䛂䝔䝄䝸䞁䜾≧ែ䛃䛸䛔䛖ືస䛜⏕䛨䜛䛣䛸䜒

㏆ᖺ䛻䛺䜚ุ᫂䛧䛶䛔䜛(24,25)(ᅗ

1-6)䚹

ᅗ1-6䠊GroEL 䛾䝔䝄䝸䞁䜾཯ᛂ䝰䝕䝹(24)

௨ୖ䜘䜚䠈GroEL 䛾ᶵ⬟䛸ᵓ㐀䛻䛚䛔䛶ヲ⣽䛻ゎᯒ䛥䜜䛶䛺䛔ኚᛶ䝍䞁䝟䜽㉁䛻ᑐ䛩䜛㔜せ䛺ᶵᵓ䛿ᮍ䛰ከ䛟ṧ䛳䛶䛔䜛䚹

(19)

11 11-3䠊䝍䞁䝟䜽㉁䛾ᵓ㐀␗ᖖ䛻䜘䜚⏕䛨䜛䜰䝭䝻䜲䝗⥺⥔จ㞟

1980

ᖺ䛻

Glenner

䛿䠈䜰䝭䝻䜲䝗䞊䝅䝇䛸䜰䝭䝻䜲䝗⥺⥔䛾㛵ಀᛶ䛻䛴䛔䛶♧䛧䛶䛔䜛(26)䚹䜰䝭䝻䜲䝗䞊䝅䝇䛸䛿䠈ᮏ᮶䠈⏕⌮ⓗᶵ⬟䜢䜒䛴䝍䞁䝟䜽㉁䛾❧యᵓ㐀䛻ኚ᥮䛜㉳䛣䜚䠈ᚤ⣽䛺

⥺⥔䛻㔜ྜ䛧䛶ỿ╔䛩䜛⑓ែ䛾⥲⛠䛷䛒䜛䚹䛣䛾䜘䛖䛺≉ᚩ䛛䜙䠈䜰䝭䝻䜲䝗䞊䝅䝇䛿䝣䜷䞊䝹䝕䜱䞁䜾⑓䛸䜒䜘䜀䜜䛶䛔䜛(27)䚹䜘䛟▱䜙䜜䛶䛔䜛䜰䝭䝻䜲䝗䞊䝅䝇䛸䛧䛶䠈䜰䝹䝒䝝䜲䝬䞊⑓䠈䝟䞊䜻䞁䝋䞁⑓䠈䝝䞁䝏䞁䝖䞁⑓䠈䝥䝸䜸䞁⑓䠈➽ⴎ⦰ᛶഃ⣴◳໬⑕(ALS)➼䛜Ꮡᅾ䛩䜛䚹䛧䛛䛧䠈䛭䛾୍᪉䛷䠈⑓Ẽ䛸㛵㐃䛧䛶䛔䛺䛔䝍䞁䝟䜽㉁䛾䜰䝭䝻䜲䝗⥺⥔໬䜒ሗ࿌䛥䜜䛶䛔䜛(28)䚹

䜰䝭䝻䜲䝗⥺⥔䛾ᙧᡂ䛿䠈ᚊ㏿ẁ㝵䛸䛺䜛᰾䜢ᙧᡂ䛩䜛䝷䜾䝍䜲䝮䠈ᙧᡂ䛥䜜䛯᰾䛻䝰䝜䝬䞊䛜ḟ䚻䛸⤖ྜ䛩䜛䛣䛸䛷⥺⥔䛜ఙ㛗䛧䛶䛔䛝䠈䛭䛾ᚋᐃᖖ≧ែ䛸䛺䜛

3

䛴䛾ẁ㝵䜢⤒䛯཯ᛂ 䛻䜘䜚⏕䛨䜛䛸䛥䜜䛶䛔䜛(ᅗ

1-7)䚹䛧䛛䛧䠈ண䜑⥺⥔䛾᩿∦䜔จ㞟άᛶ䜢♧䛩䛂Seed䛃ศᏊ䛜

Ꮡᅾ䛩䜛䛣䛸䛻䜘䜚䠈䝷䜾䝍䜲䝮䜢⤒䛪⥺⥔ఙ㛗཯ᛂ䛜⏕䛨䜛䛸䛥䜜䛶䛔䜛(29)䚹

ᅗ1-7䠊䜰䝭䝻䜲䝗⥺⥔ᙧᡂ䝰䝕䝹

(20)

12

⌧ᅾ䛾䜰䝭䝻䜲䝗⥺⥔䛾ᐃ⩏䛸䛧䛶䠈䝁䞁䝂䝺䝑䝗Ⰽ⣲

(30)䠈Thioflavin-T(31)➼䛷ᰁⰍ䛥䜜䠈

㟁Ꮚ㢧ᚤ㙾䛷ほᐹ䛩䜛䛸ᖜ⣙

7䡚12 nm

䛾ᯞศ䛛䜜䛾䛺䛔⥺⥔ᵓ㐀䜢♧䛩(32)䚹䛣䜜䛻ຍ 䛘䠈X⥺ᅇᢡ䠈෇೫ග஧Ⰽᛶゎᯒ➼䛷䃑䝅䞊䝖䜢࿊䛩䜛➼䛜ᣲ䛢䜙䜜䜛(ᅗ

1-8)(33,34)䚹

ᅗ1-8䠊䜰䝭䝻䜲䝗⥺⥔䛾䜽䝻䝇䃑ᵓ㐀䛾ᶍᘧᅗ䛸㟁Ꮚ㢧ᚤ㙾⏬ീ

A䠊䜰䝭䝻䜲䝗⥺⥔䛾ศᏊ䝰䝕䝹ᅗ B䠊䜰䝭䝻䜲䝗⥺⥔䛾㟁Ꮚ㢧ᚤ㙾⏬ീ

䜲䞁䝇䝸䞁䛻䛚䛔䛶ᙧᡂ䛥䜜䜛䜰䝭䝻䜲䝗⥺⥔䛿㟁Ꮚ㢧ᚤ㙾ほᐹ➼䛻䛚䛔䛶䠈䛭䛾ᙧ≧䛿㔪

≧䛾䜒䛾䛜ከ䛟䜏䜙䜜䜛䛜䠈㑏ඖ๣䛷䛒䜛

TCEP

Ꮡᅾୗ䛻䛚䛔䛶㓟ᛶ䞉㧗 ᮲௳ୗ䛷ᜏ 㟼

⨨䛩䜛䛸䝚䞊䝗䝹≧䛾䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛(ᅗ

1-9)䚹䛥䜙䛻䠈䛣䛾䝚䞊䝗䝹≧䛾䜒䛾䛿䠈㔪

≧䛾䜒䛾䛸ẚ䜉䛶䜋䛸䜣䛹⣽⬊ẘᛶ䛜䛺䛔䛣䛸䛜♧䛥䜜䛶䛔䜛(35)䚹

A

A ^㻌 B B ^㻌

(21)

13

ᅗ1-9䠊䜲䞁䝅䝳䝸䞁䜘䜚ᙧᡂ䛥䜜䛯䜰䝭䝻䜲䝗⥺⥔(35) 㻌 A䠊TCEP 㠀Ꮡᅾୗ䛻䛚䛔䛶ᙧᡂ䛧䛯䜰䝭䝻䜲䝗⥺⥔

B䠊TCEPᏑᅾୗ䛻䛚䛔䛶ᙧᡂ䛧䛯䜰䝭䝻䜲䝗⥺⥔

䛣䛾䜘䛖䛻䠈ᙧᡂ䛥䜜䜛䜰䝭䝻䜲䝗⥺⥔䛾ᙧែ䛻䜘䛳䛶䜒⣽⬊ẘᛶ䛻㐪䛔䛜䜏䜙䜜䜛䚹䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛䝍䞁䝟䜽㉁䛾ከ䛟䛜⚄⤒ኚᛶ⑌ᝈ䛻䛚䛔䛶㛵୚䛧䛶䛔䜛䛸䛥䜜䛶䛔䜛䛜䠈䜰䝭䝻䜲䝗⥺⥔⮬య䛻ẘᛶ䛜䛒䜛䛾䛛䠈⥺⥔ᙧᡂ㐣⛬䛻䛚䛡䜛จ㞟୰㛫య䛻ẘᛶ䛜䛒䜛䛾䛛䛿 ᮍ䛰᫂☜䛻ุ᫂䛧䛶䛔䛺䛔䚹䛭䛾䛯䜑䠈䜰䝭䝻䜲䝗⥺⥔䛻⮳䜛䜎䛷䛾จ㞟⤒㊰䛸䛭䛾㐣⛬䛷ᙧ ᡂ䛥䜜䜛จ㞟ᙧែ䛾ゎᯒ䛿䠈௒ᚋ䛾䜰䝭䝻䜲䝗◊✲䛻䛚䛔䛶㔜せ䛺せᅉ䛸䛺䜛䛣䛸䛜⪃䛘䜙䜜䜛䚹䜰䝭䝻䜲䝗⥺⥔䛾ᙧᡂ䛻䛚䛡䜛ᵝ䚻䛺᪉ἲ䛻䜘䜛จ㞟ᢚไຠᯝ䛜ሗ࿌䛥䜜䛶䛝䛶䛔䜛䚹䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛䝰䝕䝹䝍䞁䝟䜽㉁䛾୍䛴䛻

GroES

䛜䛒䜛䚹䛣䜜䜎䛷䠈ᮏ◊✲ᐊ䛷䛿䝫䝸䝣䜵䝜䞊䝹䛾୍✀䛷䛒䜛䜰䞁䝖䝅䜰䝙䞁䜢୺ᡂศ䛸䛩䜛䝡䝹䝧䝸䞊䜶䜻䝇(VMA)䜢໬Ꮫ䝅䝱䝨䝻䞁䛸䛧䛶⏝䛔䛯

GroES

䛾จ㞟ᢚไຠᯝ䛻䛴䛔䛶ሗ࿌䛧䛶䛔䜛(ᅗ

1-10)(36)䚹

㻌㻌

A

^㻌

B B

^㻌

(22)

14

ᅗ1-10䠊VMA䛻䜘䜛GroES䛾䜰䝭䝻䜲䝗⥺⥔ᙧᡂᢚไ(36) A䠊␗䛺䜛᫬㛫䛾VMAῧຍ䛻䜘䜛GroES䛾จ㞟㐣⛬䛻䛚䛡䜛⺯ගኚ໬

㻌㻌㻌 48᫬㛫(䖃)䠈96᫬㛫(䖃)䠈120᫬㛫(䖃)䛻䛚䛡䜛VMAῧຍ(ῧຍ䛿▮༳䛷⾲♧) B䠊120᫬㛫䛷䛾AFM䛻䜘䜛GroES䛾䜰䝭䝻䜲䝗⥺⥔ほ (㉥⥺䛻䛚䛡䜛▮༳䛾Ⅼ) C䠊VMAῧຍᚋ䛾AFMほ (㉥⥺▮༳䛷VMA䜢ῧຍ䛧䛶48᫬㛫᣺┞ᚋ)

䝫䝸䝣䜵䝜䞊䝹䛸䛿䝧䞁䝊䞁⎔䠈䝘䝣䝍䝺䞁⎔➼䛾ⰾ㤶᪘䛻⤖ྜ䛧䛯䝠䝗䝻䜻䝅ᇶ䜢ᣢ䛴໬ྜ

≀䛾⥲⛠䛷䠈䜹䝔䜻䞁䠈䜽䝹䜽䝭䞁䠈䝞䜲䜹䝺䞁➼䛜ᣲ䛢䜙䜜䜛䚹䜹䝔䜻䞁䛾୍✀䛷䛒䜛ἐ㣗Ꮚ 㓟䜶䝢䜺䝻䜹䝔䜻䞁䛿䝥䝸䜸䞁

PrP

^sc 䛾⥺⥔ᙧᡂ䜢㜼ᐖ䛧(37)䠈䜽䝹䜽䝭䞁䛿䜰䝭䝻䜲䝗䃑(A䃑) 䛾䜸䝸䝂䝬䞊䛸⥺⥔ᙧᡂ䜢ᢚไ䛧

(38)

䠈䝞䜲䜹䝺䞁䛿䃐

-Synuclein

䛾⥺⥔ఙ㛗䛻ᑐ䛧䛶ᢚไ

ຠᯝ䛜䛒䜛(39)䛸䛭䜜䛮䜜♧䛥䜜䛶䛔䜛䚹

䛥䜙䛻䠈ALS 䛾ཎᅉ䝍䞁䝟䜽㉁䛷䛒䜛䝇䞊䝟䞊䜸䜻䝃䜲䝗䝆䝇䝮䝍䞊䝊(SOD1)䛿䛭䛾άᛶ 㒊఩䛻

Cu

䛸

Zn

䛜άᛶ୰ᚰ䛻㓄఩䛩䜛䛣䛸䛷䠈άᛶ㓟⣲䜢ศゎ䛩䜛ാ䛝䜢♧䛩䛜䠈Cu䛾⤖

ྜ䛻䜘䜚䛭䛾จ㞟䜒ᢚไ䛥䜜䜛䛾䛜♧䛥䜜䛶䛔䜛(40)䚹䛥䜙䛻䠈䜰䝹䝒䝝䜲䝬䞊⑓䛾ཎᅉ䝍䞁䝟䜽㉁䛷䛒䜛

A䃑(41)䠈䝥䝸䜸䞁⑓䛾ཎᅉ䝍䞁䝟䜽㉁ PrP

^C^㻌

(42)䛻䛚䛔䛶䜒㔠ᒓ䜲䜸䞁䛜㛵୚䛧

(23)

15

䛶䛔䜛䛸ሗ࿌䛥䜜䛶䛔䜛䚹䝥䝸䜸䞁䝍䞁䝟䜽㉁䛜㛵୚䛧䛶䛔䜛⑌ᝈ䛸䛧䛶䝠䝖䛻䛚䛡䜛䜽䝻䜲䝒䝣䜵䝹䝖-䝲䝁䝤⑓(CJD)䛜䛒䜚䠈䜴䝅䛷䛿䜴䝅ᾏ⥥≧⬻⑕

(BSE)䛜䛒䜛䛜䠈䛣䛾䝥䝸䜸䞁䝍䞁䝟䜽㉁

䛜

A䃑䜸䝸䝂䝬䞊䛾⑓ཎస⏝䛻㛵୚䛧䛶䛔䜛䛣䛸䛜㏆ᖺ♧䛥䜜䛶䛔䜛(43)䚹ṇᖖ䛺䝬䜴䝇䛾ᾏ

㤿ษ∦䛻䛚䛔䛶䠈A䃑䜸䝸䝂䝬䞊䛿䠈LTP(㛗ᮇቑᙉ䠖䝙䝳䞊䝻䞁㛫䛾䝅䝘䝥䝇⤖ྜ䜢ቑᙉ䛩䜛

⌧㇟

)

䛜㜼ᐖ䛥䜜䛶䛔䜛䛾䛜♧䛥䜜䛶䛔䜛䛜䠈

PrP

^c

(⣽⬊ᆺ䝥䝸䜸䞁)䜢ಖᣢ䛧䛶䛔䛺䛔䝬䜴䝇䛾

ᾏ㤿ษ∦䛷䛿㜼ᐖ䛥䜜䛶䛔䛺䛛䛳䛯䚹䛣䜜䛸ྠᵝ䛻䠈ṇᖖ䝬䜴䝇䛾ᾏ㤿ษ∦䛷

A䃑䛸 PrP

^C䛾

┦஫స⏝䜢㐽᩿䛩䜛䛸䠈

LTP

䛿

A

䃑䜸䝸䝂䝬䞊䛻䜘䜛ᙳ㡪䜢ཷ䛡䛺䛛䛳䛯䛣䛸䜒♧၀䛥䜜䛯䚹䛣䜜䜘䜚䠈䝥䝸䜸䞁䛿䠈⣽⬊ෆ䛷䛾

A䃑䛾䝣䜷䞊䝹䝕䜱䞁䜾≧ែ䜢ኚື䛥䛫䜛䛂⑓ཎᛶ䝅䝱䝨䝻䞁䛃

䛸䛧䛶䛾ᶵ⬟䜢䜒䛳䛶䛔䜛ྍ⬟ᛶ䜒⪃៖䛥䜜䛶䛔䜛䚹⚄⤒ኚᛶ⑌ᝈ䛻䛚䛔䛶䛿䠈ᮍ䛰䛻☜❧䛧䛯἞⒪ἲ䛿Ꮡᅾ䛧䛶䛔䛺䛔䛜䠈ᵝ䚻䛺ᢚไᅉᏊ䛸䛾㛵㐃䛿௒ᚋ䛾἞⒪ἲ㛤Ⓨ䛻᭷ຠ䛺▱ぢ䜢୚䛘䜛䛣䛸䛜ᮇᚅ䛥䜜䛶䛔䜛䚹

1-4䠊䝟䞊䜻䞁䝋䞁⑓Ⓨ⑕䛾ཎᅉ䝍䞁䝟䜽㉁䃐-Synuclein

1817

ᖺ䛻

Parkinson

䛻䜘䜚Ⓨぢ䛥䜜䛯䝟䞊䜻䞁䝋䞁⑓䛿ᙜึ᣺ᡓ㯞⑷䛸䛧䛶ሗ࿌䛥䜜䠈䛭䛾ᚋ

1888

ᖺ䛻

Charcot

䛜䜘䜚ヲ⣽䛺⑕౛䜢ᥦ♧䛧䠈䝟䞊䜻䞁䝋䞁⑓䛸䛧䛶⤂௓䛥䜜䛯(44)䚹䝟䞊䜻䞁䝋䞁⑓(PD)䛿䠈␗ᖖ䝍䞁䝟䜽㉁䛾⵳✚䛜ཎᅉ䛷⏕䛨䜛⚄⤒ኚᛶ⑌ᝈ䛾

1

䛴䛸䛧䛶䠈

⌧ᅾ䜰䝹䝒䝝䜲䝬䞊⑓䛻ḟ䛔䛷㢖⦾䛻㉳䛣䜛⑌ᝈ䛷䛒䜛䛸䛥䜜䛶䛔䜛䚹䝟䞊䜻䞁䝋䞁⑓䛻䛚䛔䛶䠈୰⬻㯮㉁䛾䝗䞊䝟䝭䞁⚄⤒䛜ኚᛶ⬺ⴠ䛧䛯䛸䛣䜝䛻䝺䝡䞊ᑠయ(LB)䛸䜘䜀䜜䜛ᑒධయ䛜 ᙧᡂ䛥䜜䜛(ᅗ

1-11)䚹

1997

ᖺ䛻

Spillantini

䜙䛿䠈ᏙⓎᛶ䝟䞊䜻䞁䝋䞁⑓䛻䛚䛡䜛

LB

䛾⥺⥔≧୺せᵓᡂᡂศ 䛜α-Synuclein䛷䛒䜛䛣䛸䜢᫂䜙䛛䛻䛧䛯(45)䚹α-Synuclein ௨እ䛾

LB

䛾ᵓᡂᡂศ䛸䛧䛶䠈 ศᏊ䝅䝱䝨䝻䞁䛷䛒䜛αB-䜽䝸䝇䝍䝸䞁䠈HSP70䠈HSP27➼௚䛾ᵝ䚻䛺䝍䞁䝟䜽㉁䜒ᣲ䛢䜙䜜䜛

(46,47)䚹

(24)

16

ᅗ1-11䠊ච␿ᰁⰍ䛥䜜䛯䝟䞊䜻䞁䝋䞁⑓Ⓨ⑕ᝈ⪅䛾㯮㉁ษ∦(45)

A䠊䃐-Synuclein䛜ᙧᡂ䝺䝡䞊ᑠయ䜢ྵ᭷䛧䛯2䛴䛾⚄⤒✺㉳䠄▮༳䛻䜘䜚⾲グ䠅 B䠊2䛴䛾䃐-Synuclein䛜ᙧᡂ䛧䛯䝺䝡䞊ᑠయྵ᭷⚄⤒⣽⬊

C䠊⣽⬊እ䛻䛚䛡䜛䃐-Synuclein䛜ᙧᡂ䛧䛯䝺䝡䞊ᑠయ

⚄⤒ኚᛶ⑌ᝈ䛻䛚䛡䜛䃐-Synuclein 䛾㛵୚䛿䠈ᙜึ䜰䝹䝒䝝䜲䝬䞊⑓(AD)䛻䛚䛡䜛䜰䝭䝻䜲䝗⌜ ⏤ ᮶䛾䃐-Synuclein ᩿∦

(NAC)䛜኱䛝䛟㛵୚ 䛧䛶䛔䜛䛸⪃䛘䜙䜜䛶䛔䛯(48)䚹䃐- Synuclein

䛾ᵓ㐀䛿䠈SNCA 㑇ఏᏊ䛻䜘䜚䜶䞁䝁䞊䝗䛥䜜䜛䜰䝭䝜㓟

140

ṧᇶ䛛䜙䛺䜛䝍䞁䝟䜽㉁䛷䠈୧ぶ፹ᛶ䛾

N

ᮎ➃䠈≉ᐃ䛾㓄ิ䜢ᣢ䛯䛺䛔ぶỈᛶ䛾

C

ᮎ➃䠈䛭䛾㛫䛻䛿␯Ỉᛶ㡿 ᇦ䛷䠈㠀䜰䝭䝻䜲䝗ᡂศ䜢ྵ䜐จ㞟䛻㛵䛩䜛

61-95

䛾

35

䜰䝭䝜㓟ṧᇶ䛛䜙ᡂ䜛

NAC

㡿ᇦ䛸

࿧䜀䜜䜛㓄ิ䛛䜙ᵓᡂ䛥䜜䛶䛔䜛䚹䜎䛯䠈Nᮎ➃ഃ䛻䛿

KTKEGV

㡿ᇦ䛛䜙䛺䜛⧞䜚㏉䛧㓄ิ

䜢ከ䛟ྵ䜣䛷䛔䜛≉ᚩ䜢ᣢ䛳䛶䛔䜛(ᅗ

1-12)(49)䚹

ᅗ1-12䠊䃐-Synuclein䛾ᵓ㐀

A䠊䃐-Synuclein䛾䜰䝭䝜㓟㓄ิ䛾ᶍᘧᅗ㻌䕔䠖KTKEGV䛾⧞䜚㏉䛧㓄ิ䜢♧䛩䚹 B䠊䝭䝉䝹⤖ྜ䃐-Synucleinᵓ㐀(50)

A

A B B C C

㻌

(25)

17 NAC

㡿ᇦ䛻Ꮡᅾ䛩䜛␯Ỉᛶṧᇶ䜢ྵ䜣䛰㡿ᇦ䛾⨨᥮䜔䛔䛟䛴䛛䜢Ḟᦆ䛥䛫䜛䛣䛸䛷䚸䜰䝭䝻䜲䝗⥺⥔䛾ᙧᡂ䛻䛚䛔䛶ኚ໬䛜⏕䛨䜛䛣䛸䛜♧䛥䜜䛶䛔䜛(51,52)䚹䛥䜙䛻䠈NAC㡿ᇦ(1-35) ༢⊂䛾䝨䝥䝏䝗䛜ᙧᡂ䛧䛯จ㞟䛿䠈Thioflavin-S 䛻㝧ᛶ䛷䛒䜛䛣䛸䛛䜙䜽䝻䝇䃑䝅䞊䝖ᵓ㐀䜢 ᙧᡂ䛧䛶䛚䜚䠈TEM 䛾ゎᯒ⤖ᯝ䛛䜙䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛧䛶䛔䜛䛣䛸䜒♧䛥䜜䛶䛔䜛(53)䚹䜎䛯䠈

NACcore(68-78

䜰䝭䝜㓟ṧᇶ

)

䝘䝜⤖ᬗ䛾ᵓ㐀䛿䠈䛣䜜䜎䛷ᑠ䛥㐣䛞䛶ගᏛ㢧ᚤ㙾䛷䛿ほᐹ䛩䜛䛣䛸䛜ฟ᮶䛺䛔䛸䛥䜜䛶䛝䛯䛜䠈㏆ᖺ䝬䜲䜽䝻㟁Ꮚᅇᢡἲ䠄MicroED䠅䛸䛔䛖ᡭἲ䜢฼

⏝䛧䠈ᵓ㐀䛜Ỵᐃ䛥䜜䛶䛔䜛䚹䛣䛾⤖ᯝ䛷䛿䠈NACcore 䛾ᵓ㐀䛿䠈䃐-Synuclein ඲㛗䛜ᙧ ᡂ䛩䜛ཎ⥺⥔䛸㢮ఝ䛧䛯ᵓ㐀䛷䛒䜛䛾䛜♧䛥䜜䛶䛔䜛(54)䚹䜰䝭䝻䜲䝗⥺⥔䛾ᙧᡂ䛻䛚䛔䛶䠈

NAC

㡿ᇦ䛻䛚䛡䜛␯Ỉᛶṧᇶ䛿㔜せ䛺ᙺ๭䜢ᯝ䛯䛧䛶䛔䜛䛸⪃䛘䜙䜜䜛䚹䃐-Synuclein 䛾

㈇䛻ᖏ㟁䛧䛶䛔䜛

C

ᮎ➃㡿ᇦ䜢Ḟᦆ䛥䛫䜛䛣䛸䛷䜰䝭䝻䜲䝗⥺⥔䛜䜘䜚᪩䛟ᙧᡂ䛩䜛䛣䛸䛜♧䛥䜜䛶䛔䜛䚹䛣䛾⤖ᯝ䜘䜚䠈C ᮎ➃㡿ᇦ䛿䠈㔜ྜ䜢㜼ᐖ䛩䜛ຠᯝ䛜⪃䛘䜙䜜䜛(55)䚹䛧䛛䛧䠈C ᮎ

➃㡿ᇦ䛻Ꮡᅾ䛧䛶䛔䜛

129

␒┠䛾

Ser

ṧᇶ(S129)䛿䠈䜹䝊䜲䞁䜻䝘䞊䝊䊠ཪ䛿䊡䛻䜘䜚䝸䞁㓟໬䛩䜛䛣䛸䛷䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛ഴྥ䛜ቑຍ䛩䜛䛸䛔䛖⤖ᯝ䜒䛒䜛䚹LB䜢ᵓᡂ䛧䛶䛔䜛䃐-Synuclein 䛻䛚䛡䜛

S129

ṧᇶ䛿䠈90% 䛜䝸䞁㓟໬䛧䛶䛔䜛䛣䛸䛛䜙䜒䠈䝟䞊䜻䞁䝋䞁⑓䛻䛚䛡䜛䃐-Synuclein 䛾

LB

䛾ᙧᡂ䛻㛵୚䛧䛶䛔䜛䛣䛸䛜⪃䛘䜙䜜䛶䛔䜛(56)䚹

2

䛴䛾䃐-Synuclein 㑇ఏᏊኚ␗య

A53T䠈A30P

䛿᪩ᮇⓎ⑕ᆺ䛾ᐙ᪘ᛶ

PD

䛾ᙧᡂ䛻䛴䛺䛜䜛䛣䛸䛜♧䛥䜜䛯(57)䚹

in vitro

䛻䛚䛔䛶䃐-Synuclein A53T䛸

A30P

䛿䜸䝸䝂䝬䞊䠈 จ㞟୰㛫య䠈⥺⥔䜢ྠ⛬ᗘᙧᡂ䛩䜛䚹䃐-Synuclein 䛾⥺⥔ᙧᡂ㐣⛬䛻䛚䛔䛶䠈A53T ኚ

␗య䛾Ꮡᅾୗ䛷䛿ຍ㏿䛥䜜䜛୍᪉䛷䠈A30P ኚ␗యᏑᅾୗ䛷䛿⥺⥔ᙧᡂ䛻ᙳ㡪䜢ཬ䜌䛥䛺䛔䛸䛥䜜䛶䛔䜛䚹䛧䛛䛧䠈A30P ኚ␗య䛿⁐ᾮ୰䛷㠀⥺⥔ᆺ䜸䝸䝂䝬䞊䛾ᙧᡂ䛸㠀ỿẊ䝍䞁䝟䜽㉁䛾ᾘ⁛䜢ຍ㏿䛩䜛(58)䚹䜎䛯䠈A30P ኚ␗య䛿㔝⏕ᆺ䛸䛭䛾⥺⥔䛾ᵓ㐀䛻㐪䛔䛜䛒䜛䛾䛜᫂䜙䛛䛸䛺䛳䛶䛔䜛(ᅗ

1-13)(59)䚹E46K

ኚ␗య䜒䠈ᐙ᪘ᛶ䝟䞊䜻䞁䝋䞁⑓䛻㛵ಀ䛩䜛ኚ␗

ᆺ䃐-Synuclein 䛸䛥䜜䛶䛔䜛(60)䚹E46K ኚ␗య䛿䠈㈇㟁Ⲵ䛻ᖏ㟁䛧䛯䝸䝫䝋䞊䝮䜈㔝⏕ᆺ 䛾䃐-Synuclein 䜘䜚䜒⤖ྜ䛧䠈䛥䜙䛻䛣䜜䛿௚䛾

2

䛴䛾ኚ␗య䛸䛿␗䛺䛳䛶䛔䜛䛣䛸䜒♧䛥䜜

(26)

18

䛶䛔䜛䚹䜰䝭䝻䜲䝗䛾⥺⥔ᙧᡂ䛻䛚䛔䛶䠈E46K ኚ␗య䛿㔝⏕ᆺ䃐-Synuclein (WT) 䛸ẚ㍑

䛧䜘䜚᪩䛟⥺⥔䜢ᙧᡂ䛩䜛䛜䠈A53T䛸ྠ⛬ᗘ䛷䛒䜛(61)䚹䛣䜜䜙

3

䛴䛾ᐙ᪘ᛶኚ␗య䛻ຍ䛘䠈

㏆ᖺ

50

␒┠䛾

His

䜢

Gln

ṧᇶ䛻⨨᥮䛧䛯

H50Q

ኚ␗య䜒ᐙ᪘ᛶኚ␗య䛸䛧䛶Ⓨぢ䛥䜜䛯

(62)䚹䛣䛾ኚ␗య䛿䠈ⴭ䛧䛟จ㞟䜢ຍ㏿䛧䠈䃐-Synuclein

䛾䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛䚹䛥䜙䛻䠈

2

ḟඖ᰾☢Ẽඹ㬆

(NMR)

䛸෇೫ග஧Ⰽᛶ

(CD)

䝇䝨䜽䝖䝹䛻䜘䜛ᵓ㐀ゎᯒ䛻䛚䛔䛶඲యⓗ䛺

2

ḟᵓ㐀䛜ᨵኚ䛧䛺䛔䛾䛜♧䛥䜜䛶䛔䜛䚹䜎䛯䛣䛾ኚ␗య䛿

in vitro

䛻䛚䛔䛶㔠ᒓ䛸┦஫స

⏝䛧䠈䝸䞁㓟໬䛩䜛⬟ຊ䜒䛺䛔䛣䛸䛜♧䛥䜜䜛䚹䛭䛾⣽⬊ẘᛶຠᯝ䛿

WT

䛸ẚ㍑䛧䠈⣽⬊እ䛷㢧ⴭ䛻ẘᛶ䛜䛷䛶䛔䜛䜘䛖䛻䜏䜙䜜䛯(62)䚹

ᅗ1-13䠊䃐-Synuclein WT 䛸A30P⏤᮶䛾䜰䝭䝻䜲䝗⥺⥔ᵓ㐀䛾㐪䛔(59) A䠊䃐-Synuclein WT 䛜ᙧᡂ䛧䛯䜰䝭䝻䜲䝗⥺⥔

B䠊䃐-Synuclein A30P 䛜ᙧᡂ䛧䛯䜰䝭䝻䜲䝗⥺⥔

㓟໬䛻䜘䜛䝇䝖䝺䝇䛿䠈䝟䞊䜻䞁䝋䞁⑓Ⓨ⑕䛾ཎᅉ 䛻䛺䜛䛸⪃䛘䜙䜜䛶䛔䜛(63)䚹䜰䝭䝜㓟ഃ

㙐䛻䛚䛔䛶䠈Met 䛿᭱䜒㓟໬䛜⏕䛨䜔䛩䛔䚹㓟໬䛧䛯

Met

䛿䠈䜰䝭䝻䜲䝗⥺⥔ᙧᡂ䜢ᢚไ䛩䜛䚹㔠ᒓ䜲䜸䞁䛿䛣䛾ᢚไຠᯝ䛻ᑐ䛧䠈⥺⥔໬䜢ᘬ䛝㉳䛣䛩(64)䚹䃐-Synuclein 䛾จ㞟䛻䛚䛔䛶䜒䠈SOD1 䜔

A䃑䠈PrP

^sc䛾䜘䛖䛻㔠ᒓ䛜䛭䛾⥺⥔໬䛻㛵୚䛧䛶䛔䜛䛣䛸䛜♧䛥䜜䛶䛔䜛䚹

㏱㐣ᆺ㟁Ꮚ㢧ᚤ㙾(TEM)䛻䜘䜛ゎᯒ䛻䛚䛔䛶䠈Cu(䊡)䛿䠈WT 䛷䛿⣽㛗䛔䝛䝑䝖䝽䞊䜽䛾䜘䛖䛺⥺⥔䛜ᙧᡂ䛥䜜䜛䛾䛻ᑐ䛧䠈ᐙ᪘ᛶኚ␗య(A30P䠈E46K䠈A53T)䛷䛿⥺⥔䛷䛿䛺䛔䜰䝰

A

^㻌

B B

^㻌

(27)

19

䝹䝣䜯䝇䛺จ㞟䜢ᙧᡂ䛧䛶䛔䛯䚹䛭䜜䛻ᑐ䛧䠈Fe(䊢)Ꮡᅾୗ䛷䛿䠈WT䠈ྛኚ␗యඹ䛻㔠ᒓ䜲䜸䞁㠀Ꮡᅾୗ䛷ᙧᡂ䛥䜜䜛䜘䛖䛺▷䛟ኴ䛔⥺⥔䛜ᙧᡂ䛥䜜䛯(ᅗ

1-14)(65)䚹

ᅗ1-14䠊Cu(䊡)ཪ䛿Fe(䊢)Ꮡᅾୗ䛻䛚䛔䛶ᙧᡂ䛩䜛䃐-Synuclein⥺⥔ᙧែ䛾㐪䛔(65)

in vitro

䛻䛚䛔䛶䠈Trp 䛸

Cys

ṧᇶ䛜Ḟᦆ䛧䛶䛔䜛 α-Synuclein 䜢

H䈻O΃䛷✜䜔䛛䛻㓟

໬䛧䛯α-Synuclein䛿䠈䜰䝭䝜㓟㓄ิ୰䛻Ꮡᅾ䛧䛶䛔䜛

4

䛴඲䛶䛾

Met

ṧᇶ䜢㑅ᢥⓗ䛻䝇䝹䝩䜻䝅䝗໬䛩䜛䚹Met 㓟໬䛻䜘䜛 α-Synuclein ⥺⥔ᙧᡂ䛾ᢚไ䛿䠈䛭䛾⥺⥔ᙧᡂ䛧䛯

Met

䛾ᩘ䛻┦ᙜ䛩䜛(66)䚹ඛ➃䛰䛡䜢ษ䜚䛸䛳䛯α-Synuclein䝨䝥䝏䝗

1-15

䛿⭷䜢ᶍೌ䛧䛯⎔ቃ 䛻䛚䛔䛶䠈N䜰䝉䝏䝹໬䠈㐟㞳䜰䝭䞁ᆺ䛾୧᪉䛷䠈CuϨ䛿

2

䛴䛾䝦䝸䜹䝹䝨䝥䝏䝗㙐

Met1

䛸

Met5

ṧᇶ䛻⤖ྜ䛩䜛䛣䛸䛻䜘䜚㘒యᙧᡂ䛜⏕䛨䜛䛾䛜Ⓨぢ䛥䜜䛯(67)䚹䛣䛾ᵓ㐀䛻䛚䛔䛶䠈㓟໬㑏ඖ䛜Ᏻᐃ䛷䠈㓟໬䛿㉳䛣䜙䛪䠈㓟⣲Ꮡᅾୗ䛾䝗䞊䝟䝭䞁䛻䛚䛔䛶䜒㠀άᛶ䛷䛒䜛䛾䛜

♧䛥䜜䛯䚹䛣䜜䜘䜚䠈⣽⬊㉁䛾

CuϨα-Synuclein

䛸䛿␗䛺䜚䠈άᛶ㓟⣲ᐜ㔞䜢ኻ䜟䛪䠈㓟໬

཯ᛂ䜢ಁ㐍䛩䜛䛣䛸䛷⭷⤖ྜ

CuϨα-Synuclein

䛿䠈㠀άᛶ㖡䜢ῶᑡ䛥䛫䜛ᶵ⬟䛜䛒䜛䛾䛜

♧䛥䜜䛶䛔䜛䚹㔠ᒓ䠈㓟໬䝇䝖䝺䝇䛭䜜䛮䜜䛜䃐-Synuclein จ㞟䛾ᅉᏊ䛻䛺䜚䛖䜛䛜䠈⏕యෆ 䛻䛚䛔䛶䠈≉䛻⬻ෆ䛻䛿䃐-Synuclein䠈㔠ᒓ䠈㓟⣲䛔䛪䜜䜒Ꮡᅾ䛧䛶䛔䜛䛾䛷䠈㔠ᒓ䛸㓟໬

䝇䝖䝺䝇䛜ඹᙺ䛩䜛䛣䛸䛷䃐-Synuclein 䛾จ㞟䛜⏕䛨䜛䛣䛸䜒⪃䛘䜙䜜䜛䚹

(28)

20

䃐-Synuclein䛾ヲ⣽䛺ᶵ⬟䛿ᮍ䛰୙᫂䛺Ⅼ䛜ከ䛔䚹䃐-Synuclein 䛿䠈䜰䝹䝁䞊䝹䝕䝠䝗䝻䝀䝘䞊䝊䠄ADH䠅䛾จ㞟䛻䛚䛔䛶

HSP27

䛸ྠᵝ䛾ᢚไຠᯝ䜢♧䛧䛯䛣䛸䛛䜙䠈௚䛾䝍䞁䝟䜽㉁䛾จ㞟䜢ᢚไ䛩䜛䝅䝱䝨䝻䞁άᛶ䜢ಖᣢ䛧䛶䛔䜛䛣䛸䜒ሗ࿌䛥䜜䛶䛔䜛(58)䚹⚄⤒ఏ㐩≀

㉁䛾ᨺฟ䛻䛚䛔䛶䠈䝅䝘䝥䝇ᑠ⬊䛸⣽⬊⭷䛾⼥ྜ䛻㛵䜟䜛≀㉁䛷䛒䜛

SNARE

䝍䞁䝟䜽㉁䛾

」ྜయᙧᡂ䛻㝿䛧䛶ാ䛟ศᏊ䝅䝱䝨䝻䞁䛸䛧䛶

DnaJ

䝣䜯䝭䝸䞊䛻ᒓ䛧䛶䛔䜛

CSP

䃐䛜Ꮡᅾ䛩䜛(68)䚹䝬䜴䝇䛻䛚䛔䛶⮴Ṛ䛻⮳䜛

CSP䃐䛾䝜䝑䜽䜰䜴䝖䛿䠈䃐-Synuclein

䜢Ⓨ⌧䛥䛫䜛䛣䛸䛻䜘䜚䛭䛾⮴Ṛ䜢ᅇ㑊䛷䛝䜛䛸䛔䛖ሗ࿌䛛䜙䠈ᮎᲈ⚄⤒䛾ᦆയ䜢ಖㆤ䛩䜛䛸䛔䛖ᶵ⬟䜢ᣢ䛳䛶䛔䜛䛣䛸䜒♧ 䛥䜜䜛(69)䚹䜎䛯䠈䃐-Synuclein 䛿⭷ 䛻⤖ ྜ䛩䜛䛣䛸䛻䜘䜚⥺ ⥔ ᙧ ᡂ䛜ຍ ㏿䛥䜜

(70)

䠈䛭䛾 ᚋ 䃐

-Synuclein

䛾 จ 㞟䛿 ⭷ 䛾⾲㠃䛷 ⏕ 䛨䜛䛣䛸䛛䜙䠈 ⭷ 䛿 ඃ ඛ ⓗ 䛻䃐

- Synuclein

䛾䜸䝸䝂䝬䞊໬䜢ㄏᑟ䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛(71)䚹䛣䜜䛻ᑐ䛧䠈୰ᛶ䛸↓ᴟᛶ ᮲௳䛾⭷䛻ᙉ䛟⤖ྜ䛩䜛䛸⥺⥔ᙧᡂ䛜㜼ᐖ䛥䜜䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛(72)䚹䛥䜙䛻䠈㓟໬䛧䛯䝸䞁⬡㉁䛿䠈⬡㉁⃰ᗘ䛾㐪䛔䛷䃐-Synuclein 䛜ᙧᡂ䛩䜛ᙧែ䛜␗䛺䜛䛾䜢♧䛧䛶䛔䜛䚹ప

⃰ᗘ᮲௳ୗ䛻䛚䛔䛶䠈䝸䞁⬡㉁ᑠ⬊䛻⤖ྜ䛩䜛䛸㒊ศⓗ䛻䝣䜷䞊䝹䝗䛧䛶⥺⥔ᵓ㐀䜢ᙧᡂ䛩䜛䛜䠈䜘䜚㧗⃰ᗘ᮲௳䛷䛿䃐䜈䝸䝑䜽䝇ᵓ㐀䜢䛸䜛䛣䛸䛷⥺⥔ᙧᡂ䛜㜼ᐖ䛥䜜䜛(73)䚹䛣䛾䜘䛖䛻䠈 ከ䛟䛾䝍䞁䝟䜽㉁䛸䝸䞁⬡㉁⭷䛸┦஫స⏝䛩䜛䛣䛸䛛䜙䃐-Synuclein 䛿䠈ከᩘ䛾䝅䜾䝘䝹⤒㊰

䛻㛵୚䛧䛶䛔䜛䛣䛸䛜⪃䛘䜙䜜䜛䚹䛴䜎䜚䠈⣽⬊ศ໬䠈䝅䝘䝥䝇ྍረᛶ䠈⣽⬊⏕Ꮡ䠈䝗䞊䝟䝭䞁

⚄⤒ఏ㐩䛾つไ䛻䛚䛡䜛ᙺ๭䜢ᢸ䛳䛶䛔䜛䛛䜒䛧䜜䛺䛔䛣䛸䛛䜙䠈䛣䜜䜙㏻ᖖ䛾ᶵ⬟䜢ᔂቯ䛥䛫䜛䛣䛸䛜⑓ⓗ䛺ᶵᵓ䛻㛵୚䛩䜛䜘䛖䛻䛺䜛䛾䛛䜒䛧䜜䛺䛔䚹

11-5䠊ኳ↛ኚᛶ䝍䞁䝟䜽㉁䛻ᑐ䛩䜛ศᏊ䝅䝱䝨䝻䞁䛾ാ䛝

ኚᛶ䛧䛯䝍䞁䝟䜽㉁䛾䝣䜷䞊䝹䝕䜱䞁䜾䜢ㄪᩚ䛩䜛ᶵ⬟䜢ᣢ䛴ศᏊ䝅䝱䝨䝻䞁䛿䠈⣽⬊Ṛ䛻

⮳䜛㔜⠜䛺⑌ᝈ䛾ཎᅉ䛸䛺䜛䝍䞁䝟䜽㉁䛾จ㞟䜢ᢚไ䛩䜛స⏝䜢♧䛩䛣䛸䜒ሗ࿌䛥䜜䛶䛔䜛䚹

║䛾䝺䞁䝈䛻䛚䛔䛶䠈ከ㔞䛻Ꮡᅾ䛧䛶䛔䜛䜽䝸䝇䝍䝸䞁䛸࿧䜀䜜䜛䝍䞁䝟䜽㉁䛿䠈จ㞟䛾ཎᅉ䜔 ᶵᵓ䛿䜎䛰ヲ⣽䛻ゎ᫂䛥䜜䛶䛔䛺䛔䛜䠈ⓑෆ㞀䜈䛾ከ኱䛺㛵୚䛜⪃៖䛥䜜䛶䛔䜛䚹䃐䜽䝸䝇䝍

(29)

21

䝸䞁䛿㒊ศⓗ䛻ኚᛶ䛧䛯䃑-䠈䃒-䜽䝸䝇䝍䝸䞁䛻ᑐ䛧䠈䝅䝱䝨䝻䞁ᶵ⬟䜢ᣢ䛴䛣䛸䛜▱䜙䜜䛶䛔䜛

(74)䚹䜎䛯䠈䃐B

䜽䝸䝇䝍䝸䞁䛿⣽⬊ෆ䛻䛚䛔䛶䜰䝹䝒䝝䜲䝬䞊⑓(75)䜔䜽䝻䜲䝒䝣䜵䝹䝖-䝲䝁䝤

⑓(76)䛺䛹䝭䝇䝣䜷䞊䝹䝕䜱䞁䜾⑓䛻㛵୚䛧䛶䛔䜛௚䛾䝍䞁䝟䜽㉁䛾จ㞟䜢㜼ᐖ䛧䛶䛔䜛䛣䛸䜒 ሗ࿌䛥䜜䛶䛔䜛䚹HSP70/HSP40 䛸

HSP104

䛿༠ྠ䛧䛶ኚᛶ䛧䛶䛔䜛䝍䞁䝟䜽㉁䜢ྍ⁐໬䛥䛫䜛䝅䝱䝨䝻䞁ຠᯝ䜢♧䛩

(77)

䚹

HSP104

䛿䠈㓝ẕ䝥䝸䜸䞁䝍䞁䝟䜽㉁

Sup35

䛾จ㞟䛾ᙧᡂ 䛻䛚䛔䛶㛵୚䛧䛶䛚䜚䠈HSP104 䛾Ⓨ⌧㔞䛜㏻ᖖ䛾䛸䛝䛿จ㞟యᙧᡂ䜢ಁ㐍䛩䜛䛜䠈㐣๫㔞䜒䛧䛟䛿Ⓨ⌧ᢚไ᫬䛻䛿ᢚไ䛾᪉ྥ䛻ാ䛟䛣䛸䛜♧䛥䜜䛶䛔䜛(78)䚹HSP70 䛸

HSP90

䛿

A

䃑1-40 䛾䜰䝭䝻䜲䝗⥺⥔ᙧᡂ䜢ᢚไ䛩䜛䛣䛸䛜♧䛥䜜䛶䛔䜛(79)䚹HSP90 䛿䠈䃐-Synuclein 䛾䜰䝭䝻䜲䝗⥺⥔໬䛻ᑐ䛧䠈ATP 㠀Ꮡᅾୗ䛷䛿䠈䜸䝸䝂䝬䞊䛜⵳✚䛩䜛䛜䠈ATP Ꮡᅾୗ䛷䜰䝭䝻䜲䝗⥺⥔䛜ᙧᡂ䛩䜛䛸䛥䜜䛶䛔䜛(80)䚹䛥䜙䛻䠈䃐-Synuclein A53T ኚ␗య䜢⏝䛔䛯ᐇ㦂䛻䛚䛔䛶䠈HSP90 䛿䃐-Synuclein 䛾ẘᛶ䜢♧䛩䜘䛖䛻䛺䜛䜸䝸䝂䝬䞊䛸ᙉᅛ䛺」ྜయ䜢ᙧᡂ 䛩䜛䛣䛸䛷ẘᛶ䜢ゎᾘ䛩䜛䛣䛸䛜♧䛥䜜䛶䛔䜛(ᅗ

1-15)(81)䚹

ᅗ1-15䠊HSP90䛻䜘䜛䃐-Synuclein䜸䝸䝂䝬䞊䛾↓ẘ໬䛾ᶵᵓ(81) (a) HSP90䛸䜸䝸䝂䝬䞊䛾┦஫స⏝ᶵᵓ

(b)HSP90䛸䃐-Synuclein䛾┦஫స⏝䛻䜘䜛䜸䝸䝂䝬䞊ẘᛶ䛾ゎᾘᶵᵓ

(30)

22 HSP70

䛿䠈⚄⤒⣽⬊䛻䛚䛔䛶ᙉไⓎ⌧䛥䛫䜛䛸

A䃑䝨䝥䝏䝗䛻䜘䜛⚄⤒ẘᛶ䜢㍍ῶ䛩䜛䛣

䛸䛜䛷䛝䜛䛾䛜♧䛥䜜䛶䛔䜛(82)䚹HSP70 䛻䛚䛔䛶䜒䠈α-Synuclein 䛾จ㞟ᢚไ䛻ാ䛟䛣䛸䛜

☜ㄆ䛥䜜䛶䛚䜚(83)䠈䛥䜙䛻䠈䛭䛾┦஫స⏝䛻䛿

N

ᮎ➃䛻Ꮡᅾ䛩䜛

ATP

ຍỈศゎ䝗䝯䜲䞁䛿 ᚲせ䛷䛿䛺䛟䠈䜐䛧䜝ᇶ㉁⤖ྜ䝗䝯䜲䞁䛰䛡䛷䜒┦஫స⏝䛧䠈จ㞟䛾ᢚไ䛻ാ䛟䛸䛥䜜䛶䛔䜛

(

ᅗ

1-16)(84)

䚹䛣䛾䛣䛸䛛䜙䠈䝍䞁䝟䜽㉁䛾จ㞟ᢚไຠᯝ䛻䛚䛔䛶䠈ศᏊ䝅䝱䝨䝻䞁䛿඲㛗䛸䛧䛶ാ䛟䛾䛷䛿䛺䛟䠈䜐䛧䜝≉ᐃ䛾㒊఩䛜㔜せ䛸䛺䜛䛣䛸䛜⪃䛘䜙䜜䜛䚹

ᅗ1-16䠊HSP70䛾ᇶ㉁⤖ྜ䝗䝯䜲䞁䛻䜘䜛䃐-Synuclein䛾䜰䝭䝻䜲䝗⥺⥔ᙧᡂᢚไຠᯝ(84) A䠊HSP70 䛾䜰䝭䝜㓟㓄ิ䛾ᶍᘧᅗ

B䠊HSP70Ꮡᅾୗ䛻䛚䛡䜛䃐-Synuclein 䛜ᙧᡂ䛧䛯䜰䝭䝻䜲䝗⥺⥔䛾TEM⏬ീ

ᕥ䠖䃐-Synuclein 䛜ᙧᡂ䛧䛯䜰䝭䝻䜲䝗⥺⥔

୰ኸ䠖HSP70/ATPᏑᅾୗ䛻䛚䛡䜛䃐-Synuclein 䛜ᙧᡂ䛧䛯䜰䝭䝻䜲䝗⥺⥔

ྑ䠖ATPຍỈศゎ㡿ᇦ㠀Ꮡᅾୗ䛾HSP70䜢క䛳䛯䃐-Synuclein ᙧᡂ䜰䝭䝻䜲䝗⥺⥔

A A

㻌

B

^㻌

(31)

23

୍᪉䠈

in vitro

䛻䛚䛔䛶䠈GroEL䛾䜰䝢䜹䝹䝗䝯䜲䞁༢⊂䛷䜒

Rhodanese䠈cyclophilin A

䛾䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ䛜♧䛥䜜䛶䛔䜛(85)䚹䛥䜙䛻䠈䜾䝹䞊䝥䊡ᆺ䝅䝱䝨䝻䝙䞁䛿䠈䜰䝢䜹䝹䝗䝯䜲䞁䛾ඛ➃䛻Ꮡᅾ䛧䛶䛔䜛

Helical protrusion

䛻䜘䜚㓝ẕ

Sup35NM

䜈⤖ྜ䛧䠈䛭䛾จ 㞟䜢ᢚไ䛧䛶䛔䜛䛣䛸䛜♧䛥䜜䛶䛔䜛(86)䚹ྠᵝ䛻䜾䝹䞊䝥䊠ᆺ䝅䝱䝨䝻䝙䞁

GroEL

䛻䛚䛔䛶䠈༢㞳䛧䛯䜰䝢䜹䝹䝗䝯䜲䞁䛿㏱ᯒ䜰䝭䝻䜲䝗䞊䝅䝇䛾ཎᅉ䛸䛥䜜䛶䛔䜛䃑䈻

-

䝭䜽䝻䜾䝻䝤䝸䞁䛾 จ㞟䜢ⴭ䛧䛟ᢚไ䛩䜛ຠᯝ䜢ᣢ䛴䝭䝙䝅䝱䝨䝻䞁䛷䛒䜛䛣䛸䛜♧၀䛥䜜䛶䛔䜛(ᅗ

1-17)(87)䚹䜰

䝢䜹䝹䝗䝯䜲䞁䛿ᇶ㉁䝍䞁䝟䜽㉁䛾ㄆ㆑䛸⤖ྜ䜢⾜䛔䠈䛭䛾ᚋ䝁䝅䝱䝨䝻䞁䛸༠ྠ䛩䜛䛣䛸䛷䝣䜷䞊䝹䝕䜱䞁䜾䛾ㄪᩚ䜢⾜䛳䛶䛔䜛䛜䠈䛣䜜䛿䠈䛩䛷䛻▱䜙䜜䛶䛔䜛ᇶ㉁䝍䞁䝟䜽㉁䛰䛡䛷䛿䛺䛟䠈ኳ↛ኚᛶ䝍䞁䝟䜽㉁䜒⤖ྜ䛩䜛䛣䛸䛜䛷䛝䠈䛭䛾จ㞟䛥䛘ᢚไ䛧䛶䛔䜛ྍ⬟ᛶ䜒⪃䛘䜙䜜䜛䚹

ᅗ1-17䠊GroEL䛾䜰䝢䜹䝹䝗䝯䜲䞁䛻䜘䜛䃑䈻-䝭䜽䝻䜾䝻䝤䝸䞁䛾⥺⥔ᙧᡂᢚไ(87)

A䠊䜰䝢䜹䝹䝗䝯䜲䞁Ꮡᅾୗ(䕿)ཪ䛿㠀Ꮡᅾୗ(䕕)䛻䛚䛡䜛䃑䈻-䝭䜽䝻䜾䝻䝤䝸䞁⥺⥔ᙧᡂ B-D䠊䃑䈻-䝭䜽䝻䜾䝻䝤䝸䞁䠖䜰䝢䜹䝹䝗䝯䜲䞁䠙1䠖0䠈1䠖2䠈1䠖1䛾⥺⥔ᙧᡂ཯ᛂ䛾TEM⏬ീ

E䠊䃑䈻-䝭䜽䝻䜾䝻䝤䝸䞁䠖GroEL=1䠖1䛻䛚䛡䜛⥺⥔ᙧᡂ཯ᛂ䛾TEM⏬ീ

(32)

24 11-6䠊ᮏ◊✲䛾┠ⓗ

ᮏ◊✲䛷䛿䝅䝱䝨䝻䝙䞁

GroEL

䛻䜘䜛䜰䝭䝻䜲䝗⥺⥔ᙧᡂ䛜⏕䛨䜛ኚᛶ䝍䞁䝟䜽㉁䛾ᢚไ ᶵᵓ䜢ᕤᏛⓗᡭἲ䛻䜘䜚ゎ᫂䛩䜛䛣䛸䜢┠ⓗ䛸䛧䛯䚹ᮏᏛ఩ㄽᩥ䛿䠈6❶䛷ᵓᡂ䛥䜜䜛䚹

➨

1

❶䛆ᗎㄽ䛇䛷䛿䠈ᮏ◊✲䛾⫼ᬒ䛸䛧䛶⚄⤒ኚᛶ⑌ᝈ䛾Ⓨ⑕ཎᅉ䛸䛺䜛ኳ↛ኚᛶ䝍䞁䝟䜽㉁䜢ᢚไ䛩䜛ᗈ⩏䛾ศᏊ䝅䝱䝨䝻䞁䠈䜎䛯ᮏㄽᩥ䛾┠ⓗ䛚䜘䜃ᵓᡂ䛻䛴䛔䛶㏙䜉䛯䚹

➨

2

❶䛆GroEL Gly192 ኚ␗య䜢⏝䛔䛯䝅䝱䝨䝻䝙䞁䛾ᶵ⬟ゎᯒ䛇䛷䛿䠈GroEL G192X ኚ␗య䜢⏝䛔䛯ᵓ㐀Ᏻᐃᛶ䠈䝅䝱䝨䝻䝙䞁ᶵ⬟䛾ゎᯒ䠈GroES 䛸䛾┦஫స⏝䛻䛴䛔䛶ホ౯ 䛩䜛䛣䛸䛷䠈Gly192 ṧᇶ䛾⨨᥮䛻䜘䜚

GroEL

䛾ᶵ⬟䛻䛹䛾䜘䛖䛺ᙳ㡪䜢୚䛘䜛䛾䛛䜢ㄪ䜉䛯䚹

➨

3

❶ 䛆

GroEL Gly192

ኚ ␗ య 䛻䜘䜛䜰䝭䝻䜲䝗⥺ ⥔ จ 㞟䛾ᢚ ไ ຠ ᯝ 䛇䛷䛿䠈㔝 ⏕ ᆺ

GroEL

ཬ䜃

G192N䠈I䠈W

ኚ␗య䜢⏝䛔䛶

A䃑䝨䝥䝏䝗䛸䃐-Synuclein

䜈䛾┦஫స⏝䠈䃐-Synuclein 䛾⥺⥔໬䛻䛚䛡䜛ᙳ㡪䛻䛴䛔䛶ホ౯䛩䜛䛣䛸䛷䠈Gly192ṧᇶ䛾⨨᥮䛻䜘䜛䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛䝍䞁䝟䜽㉁䜈䛾ຠᯝ䛻䛴䛔䛶ㄪ䜉䛯䚹

➨

4

❶䛆䝭䝙䝅䝱䝨䝻䞁䛂GroEL-AD䛃䜢⏝䛔䛯䜰䝭䝻䜲䝗⥺⥔ᙧᡂᢚไຠᯝ䛇䛷䛿䠈GroEL 䛻䛚䛡䜛䜰䝢䜹䝹䝗䝯䜲䞁䛜䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛䝍䞁䝟䜽㉁䛻ᙳ㡪䛧䛶䛔䜛䛣䛸䛜♧၀䛥䜜䛯䛾䛷䠈䜰䝢䜹䝹䝗䝯䜲䞁༢⊂䛻䜘䜛䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛䝍䞁䝟䜽㉁䜈䛾ຠᯝ䜢ㄪ䜉䛯䚹

➨

5

❶䛆⥲ྜ⪃ᐹ䛇䛷䛿䠈GroEL G192X ኚ␗య䛸

GroEL

䛾䜰䝢䜹䝹䝗䝯䜲䞁䛜䜰䝭䝻䜲䝗

⥺⥔䜢ᙧᡂ䛩䜛䝍䞁䝟䜽㉁䛻ᑐ䛩䜛ᙳ㡪䛾⤖ᯝ䜢⥲ྜ⪃ᐹ䛧䛯䚹

➨

6

❶䛆⤖ㄒ䛇䛷䛿䠈ᮏ◊✲䛾䜎䛸䜑䜢㏙䜉䛯䚹

(33)

25 ➨

➨ 2 ❶ GroEL Gly192 ኚ ␗ య 䜢 ⏝ 䛔䛯䝅䝱䝨䝻䝙䞁㻌㻌㻌㻌㻌 ᶵ⬟䛾ゎᯒ

2-1䠊⥴ゝ

䝍䞁䝟䜽㉁䛿ᅛ᭷䛾❧యᵓ㐀䛻䝣䜷䞊䝹䝕䜱䞁䜾䛥䜜䜛䛣䛸䛻䜘䜚ᮏ᮶䛾ᶵ⬟䜢Ⓨ᥹䛩䜛䚹䛧䛛䛧䠈ᵝ䚻䛺せᅉ䛻䜘䜚Ᏻᐃ䛺ᵓ㐀䛷䛿䛺䛔䝍䞁䝟䜽㉁䜒⏕యෆ䛻ከᩘᏑᅾ䛩䜛䚹䛣䜜䜙䛾䝍䞁䝟䜽㉁䜢ᮏ᮶䛾ᵓ㐀䛻ṇ䛧䛟䝸䝣䜷䞊䝹䝕䜱䞁䜾䛩䜛ᶵ⬟䜢䝅䝱䝨䝻䝙䞁

GroEL

䛿ಖᣢ䛧䛶䛔䜛䚹GroEL䛾ᵓ㐀䛸ᶵ⬟䛻䛿ᐦ᥋䛺㛵ಀ䛜ぢ䜙䜜䜛䚹GroEL䛻Ꮡᅾ䛩䜛䜰䝭䝜㓟ṧᇶ䜢 ኚ໬䛥䛫䜛䛣䛸䛷ᮏ᮶䛾ᶵ⬟䜢኱䛝䛟ᨵኚ䛩䜛䛣䛸䛜▱䜙䜜䛶䛔䜛䚹GroEL䝃䝤䝴䝙䝑䝖䛻䛚䛡䜛䜶䜽䜰䝖䝸䜰䝹䝗䝯䜲䞁䛿䠈䝚䜽䝺䜸䝏䝗䛾⤖ྜཬ䜃ຍỈศゎ䛾ᶵ⬟䜢᭷䛧䛶䛔䜛(9)䚹䛣䛾䝗䝯䜲䞁䛻Ꮡᅾ䛩䜛

398

␒┠䛾

Asp

ṧᇶ䜢

Ala

䛻⨨᥮䛧䛯

D398A

ኚ␗య䛿䠈GroEL䛜ᮏ᮶ ಖᣢ䛧䛶䛔䜛

ATP

䛾ຍỈศゎ⬟ຊ䛜኱ᖜ䛻ῶᑡ䛩䜛䛣䛸䛜▱䜙䜜䛶䛔䜛(21)䚹ᮏ◊✲ᐊ䛻䛚䛔䛶䜒䜶䜽䜰䝖䝸䜰䝹䝗䝯䜲䞁䛻Ꮡᅾ䛩䜛

138

␒┠䛾

Cys

ṧᇶ䜢

Trp

䛻⨨᥮䛧䛯

C138W

ኚ

␗య䛿䠈25Υ䛸

37Υ䛾␗䛺䜛 ᗘ䛷䝅䝱䝨䝻䝙䞁䝃䜲䜽䝹䛜ኚ໬䛧䛶䛔䜛䛾䜢ሗ࿌䛧䛯(11)䚹

GroEL

䛜ᮏ᮶ಖᣢ䛧䛶䛔䜛ᶵ⬟䛾䜘䜚ヲ⣽䛺ゎᯒ䜢⾜䛖䛻䛒䛯䜚䠈≉ᐃ䛾㒊఩䛻䛚䛡䜛䜰䝭䝜

㓟ṧᇶ䜢⨨᥮䛩䜛䛣䛸䛿㔜せ䛺ᡭἲ䛸䛺䜚䛖䜛䚹➨

1

❶䛷㏙䜉䛯䜘䛖䛻䠈Hinge䊡㒊఩䛻Ꮡᅾ 䛩䜛

Gly192

䜢

Trp

䛻⨨᥮䛧䛯

GroEL G192W

ኚ␗య䛾䝅䝱䝨䝻䝙䞁䛸䛧䛶䛾ᶵ⬟䜢ゎᯒ䛧䛯䛸䛣䜝

GroEL WT

䛸ẚ㍑䛧䠈኱䛝䛟άᛶ䛜పୗ䛧䛶䛔䛯䚹䛭䛣䛷䠈ᮏ❶䛷䛿䠈䜰䝭䝜㓟ṧᇶ䛾

኱䛝䛥䜢⾲䛩ᣦᶆ䛷䛒䜛䝣䜯䞁䝕䝹䝽䞊䝹䝇య✚䛻ὀ┠䛧䠈Gly192 䜢ᵝ䚻䛺኱䛝䛥䛾䜰䝭䝜㓟 ṧᇶ䛻⨨᥮䛧䛯

GroEL G192X

ኚ␗య䜢స〇䛧䛶᭦䛻ヲ⣽䛺ᶵ⬟ゎᯒ䜢⾜䛳䛯䚹GroEL

G192X

ኚ␗య䜢⏝䛔䛶ᵓ㐀Ꮫⓗ䛺㐪䛔䠈䝅䝱䝨䝻䝙䞁ᶵ⬟䜈䛾ᙳ㡪䠈ATP㠀Ꮡᅾୗ䛻䛚䛡

䜛

GroES

䛸䛾┦஫స⏝䜢ㄪ䜉䠈Gly192 ṧᇶ䛾ኚ໬䛻క䛖ᵓ㐀䞉ᶵ⬟䛻䛴䛔䛶ホ౯䛧䛯䚹䛭䛣䛷䠈Gly192ṧᇶ䛾

GroEL

䛻䛚䛡䜛䝅䝱䝨䝻䝙䞁ᶵ⬟䜈䛾㛵୚䜢㆟ㄽ䛩䜛䚹

(34)

26 22-2䠊ᮦᩱ䛸᪉ἲ

2-2-1

䠊䝥䝷䝇䝭䝗༢㞳

┠ⓗ䛾䝥䝷䝇䝭䝗䜢ಖᣢ䛧䛶䛔䜛኱⭠⳦䛾䜾䝸䝉䝻䞊䝹䝇䝖䝑䜽䜢

LB

ᇵᆅ (Ampicillin 50 μg/ml) 䛻᳜ ⳦ 䛧䠈

37Υ

䛷㟼 ⨨ ᇵ 㣴䛧䛯䚹ḟ 䛻䠈䛣䛾ᇵ 㣴 ᾮ

3 ml

䜢㞟 ⳦ 䛧䠈illustra^TM

plasmid Prep mini spin kit

䜢⏝䛔䛶䝥䝷䝇䝭䝗䜢༢㞳䛧䛯䚹

Milli-Q

䛻䜘䜚⁐ฟ䛧䛯ᚋ䠈

Nano Vue (GE Healthcare)

䜢౑⏝䛧䠈260 nm䛾྾ගᗘ䜢 ᐃ䛧䛶䝥䝷䝇䝭䝗⃰ᗘ䜢ồ䜑䛯䚹DNA䛾⃰ᗘィ⟬䛿ග㊰㛗

10 mm䠈྾ගᗘ 1

䛾䛸䛝䠈50 μg/ml㻌䛸䛺䜛䛣䛸䜢฼⏝䛧䛯䚹

2-2-2䠊䜰䜺䝻䞊䝇㟁ẼὋື

㻌䜰䜺䝻䞊䝇

S

䜢

1.0 %

䠄w/v䠅䛻䛺䜛䜘䛖䛻

TAE Buffer

䛻ຍ䛘䠈㟁Ꮚ䝺䞁䝆䛷ຍ⇕䛧䠈䜰䜺䝻䞊䝇䜢᏶඲䛻⁐䛛䛧䛯䚹ே⫙䛻䛺䜛䜎䛷෭䜎䛧䠈䜶䝏䝆䜴䝮䝤䝻䝬䜲䝗䜢

0.1

μg/ml 䛻䛺䜛䜘䛖䛻ຍ䛘䠈䝀䝹స〇ྎ䛻ὀ䛞䝁䞊䝮䜢䛥䛧䛶ᐊ 䛷ᅛ໬䛧䛯䚹ᅛ໬䛧䛯䜰䜺䝻䞊䝇䝀䝹䜢Ὃື

ᵴ䛻䝉䝑䝖䛧䠈Ὃື⏝䛾

TAE Buffer

䜢ὀ䛞䠈䝃䞁䝥䝹䜢䝀䝹ୖ䛾䜴䜵䝹䛻䜰䝥䝷䜲䛧䛯䚹㏻㟁 ᚋ䠈┠ⓗ䛸䛩䜛

BPB

䛜䝀䝹䛾

2/3

⛬ᗘ䜎䛷ὶ䜜䛯᫬Ⅼ䛷Ὃື䜢䜔䜑䠈trans䜲䝹䝭䝛䞊䝍䞊

(FAS-ϩ䠈TOYOBO)䛻䜘䜚䝞䞁䝗䜢☜ㄆ䛧䛯䚹

䠷TAE Buffer䠹

40 mM Tris-HCl(pH8.0 at 25Υ) 40 mM CH

3

COOH

1.0 mM EDTA

䠷LB ᇵᆅ⤌ᡂ䠹

1.0 %(w/v)Bacto Tryptone

0.5 %(w/v)Bacto Yeast Extract

1.0 %(w/v)NaCl

(35)

27 22-2-3

䠊ᙧ㉁㌿᥮

䝥䝷䝇䝭䝗

DNA (100-200 ng)

䜢䝁䞁䝢䝔䞁䝖䝉䝹(JM109(DE3)ᰴ

100

μl ཪ䛿

BLR (DE3)ᰴ 200 μl)䛻ຍ䛘䠈ị୰䛷 30

ศ䜲䞁䜻䝳䝧䞊䝖䛧䛯䚹ḟ䛻

42Υ䛷 45

⛊㛫ಖ 䛩䜛䛣䛸䛷䝠䞊䝖䝅䝵䝑䜽䜢⾜䛔䠈䛭䛾ᚋ┤䛱䛻ị୰䛻䛶

2

ศ㛫㟼⨨䛧䠈ண䜑

42Υ䛻䜑䛶䛚䛔䛯 SOC

ᇵᆅ䜢඲㔞䛜

1 ml

䛻䛺䜛䜘䛖䛻ຍ䛘䠈

37

Υ䛷

1-2

᫬㛫᣺┞䛧䛯䚹ᇵ㣴⁐ᾮ䜢

LB

ᐮኳᇵᆅ

(Ampicillin 50 μg/ml)䛻ሬᕸ䛧䠈37

Υ䛷୍ᬌ㟼⨨ᇵ㣴䛧䛯䚹

2-2-4

䠊

DNA Sequence

┠ⓗ㑇ఏᏊ䛾ቑᖜ䛿䠈BigDye^®

Terminator ver 3.1 Cycle Sequencing Kit (Applied Biosystems)䜢⏝䛔䠈PCR

䛻䜘䜚┠ⓗ㓄ิ䛾ቑᖜ䜢⾜䛳䛯䚹PCRᚋ䠈125 mM EDTA 5 μl䠈

Milli-Q

Ỉ

10

μl䠈䛥䜙䛻

99.5 %

䜶䝍䝜䞊䝹

60

μl䜢ຍ䛘䠈ᐊ 䛷

15

ศ㛫㟼⨨䛧䛯䚹4䉝䠈

15,000 rpm䠈30

ศ㛫㐲ᚰ䛧䛶Ὑί䛧䛯䚹㐲ᚰ⤊஢ᚋ䛻ୖΎ䜢㝖ཤ䛧䠈70 % 䜶䝍䝜䞊䝹

60

μl 䜢ຍ䛘䠈4䉝䠈15,000 rpm䠈30 ศ 㛫㐲 ᚰ 䛧䛶Ὑ ί䛧䛯䚹㐲 ᚰ ⤊ ஢ᚋ䛻 ୖ Ύ䜢㝖 ཤ 䛧䠈

Milli-Q

Ỉ

25 μl

䜢ຍ䛘䛶䝪䝹䝔䝑䜽䝇䛧䛯䚹㐽ග䛧䛯≧ែ䛷෭ⶶಖᏑ䛧䠈㫽ྲྀ኱Ꮫ⏕࿨ᶵ⬟

◊✲ᨭ᥼䝉䞁䝍䞊䛻䝅䞊䜽䜶䞁䝇ゎᯒ䜢౫㢗䛧䛯䚹䠷SOCᇵᆅ(pH7.0)䠹

2.0 %(w/v) Bacto Tryptone 0.5 %(w/v) Bacto Yeast Extract 10 mM NaCl

2.5 mM KCl 10 mM MgCl΃

10 mM MgSO΅

20 mM Glucose

(36)

28 22-2-5

䠊

GroEL G192X

䛾స〇

䝅䝱䝨䝻䝙䞁

GroEL(DR-1

䠈SR-1) G192X ྛ ኚ ␗ య䛿䠈GroEL(DR-1)Ⓨ ⌧䝥䝷䝇䝭䝗

pETEL

ཪ䛿

SR-1

Ⓨ⌧䝥䝷䝇䝭䝗

pEL SR-1

䜢㗪ᆺ䛸䛧䠈PCR䜢⏝䛔䛯Ⅼኚ␗ᑟධ䛻䜘䜚స

〇䛧䛯䚹ྛኚ␗య䛾䝥䝷䝇䝭䝗

pETEL G192X

ཪ䛿

pEL SR-1 G192X

䜢⏝䛔䛶኱⭠⳦

JM109

䜢ᙧ㉁㌿᥮䛧䠈䛭䛾ᚋ

DNA Sequence

䛻䜘䜚ኚ␗ᑟධ䜢☜ㄆ䛧䛯䚹

2-2-6

䠊䝍䞁䝟䜽㉁ᐃ㔞

5

ಸᕼ㔘䛧䛯

Bio-Rad

♫〇

Protein assay kit

䜢᳨㔞⥺స〇⏝ヨ㦂⟶

6

ᮏ䛸䝃䞁䝥䝹 ᐃ⏝䛾ヨ㦂⟶䛻ྛ

2ml

䛪䛴ศὀ䛧䛯䚹᳨㔞⥺⏝ヨ㦂⟶䛻┠ⓗ䛾⃰ᗘ䛸䛺䜛䜘䛖䛻

BSA(0.5 mg/ml)䛸 Milli-Q

Ỉ䜢ୗグ䛾䜘䛖䛻ຍ䛘䠈᳨㔞⥺䛾స〇䜢⾜䛳䛯䚹

⃰ᗘ (mg/ml)

0.0 0.1 0.2 0.3 0.4 0.5 BSA (μl)

Milli-Q

0.0 40

8.0 32

16 24

24 16

32 8.0

40 0.0

ḟ䛻䝃䞁䝥䝹䜢㐺ᐅᕼ㔘䛧䛶

0~0.5 mg/ml

䛾⠊ᅖ䛻཰䜎䜛䜘䛖䛻䝃䞁䝥䝹 ᐃ⏝䛾ヨ㦂

⟶䛻ຍ䛘䠈䝃䞁䝥䝹඲㔞䛜

40

䃛l䛻䛺䜛䜘䛖䛻

Milli-Q

Ỉ䜢ຍ䛘䛯䚹ḟ䛻

595 nm

䛷䛾྾ග ᗘ䜢 ᐃ䛧䠈ẚⰍᐃ㔞䛻䜘䜚⃰ᗘ䜢ồ䜑䛯䚹

2-2-7

䠊኱㔞ᇵ㣴

LB

ᇵᆅ䜢⏝䛔䠈኱㔞ᇵ㣴䜢⾜䛳䛯䚹඲䛶䛾ᇵ㣴䛿

37䉝䛷⾜䛳䛯䚹ᇵᆅ⤌ᡂ୪䜃䛻ᡭ㡰

䜢௨ୗ䛻♧䛧䛯䚹

(37)

29

⳦య䛿ᙧ㉁㌿᥮ᚋ䠈LB/Ampᐮኳᇵᆅ䛷

Over night

㟼⨨ᇵ㣴䛧䛯䚹䝅䞁䜾䝹䝁䝻䝙䞊䜢 ༢㞳䛧䠈5 ml䛾

LB/Amp

ᾮయᇵᆅ (ヨ㦂⟶) 䛻᳜⳦䛧䠈OD600䍦

0.6

䛻䛺䜛䜎䛷᣺┞ᇵ㣴䛧䛯䚹ḟ䛻

1䊬䛾䝞䝑䝣䝹௜䛝୕ゅ䝣䝷䝇䝁䛻ῧຍ䛧䛯 300 ml

䛾

LB/Amp

ᾮయᇵᆅ䛻ヨ㦂

⟶䛷ᇵ㣴䜢⾜䛳䛯ᇵ㣴⁐ᾮ

300

μl 䜢᳜⳦䛧䠈37䉝䠈120 rpm 䛷᣺┞ᇵ㣴䛧䛯䚹

OD

600 䍦

0.4-0.6

䛻䛚䛔䛶⤊⃰ᗘ

1 mM

䛸䛺䜛䜘䛖䛻

IPTG

䜢ῧຍ䛧䠈෌䜃

5

᫬㛫᣺┞ᇵ㣴䛧䛯䚹ᇵ 㣴⤊஢ᚋ䠈8000 rpm䠈20 ศ䠈4 䉝䛷㐲ᚰศ㞳䛻䜘䜚㞟⳦䛧䛯䚹

2-2-8

䠊

GroEL(

㔝⏕ᆺ䠈ྛ✀ኚ␗య

)

䛾⢭〇

GroEL

㔝 ⏕ ᆺ ཪ 䛿 ྛ ✀ ኚ ␗ య 䛾 ⢭ 〇䛿䠈 ᐊ 䛻䛶

AKTA-FPLC system(GE Healthcare)䜢⏝䛔䛯 Superdex 200 Increase 10/300 GL(GE Healthcare)䛻䜘䜛䝀䝹䜝

㐣䜽䝻䝬䝖䜾䝷䝣䜱䞊䠈⥆䛔䛶

Resource-Q (GE Healthcare)

䛻䜘䜛㝜䜲䜸䞁஺᥮䜽䝻䝬䝖䜾䝷䝣䜱䞊䜢⾜䛳䛯䚹⢭〇䛻⏝䛔䛯

Buffer

䛸ᡭ㡰䜢௨ୗ䛻♧䛧䛯䚹

―౑⏝

Buffer䠉

䠷LBᾮయᇵᆅ䠄pH7.4䠅䠹

Polypepton 1.0%

Yeast Extract 0.5%

NaCl 1.0%

䠷LB ᐮኳᇵᆅ䠄pH7.4䠅䠹

Polypepton 1.0%

Yeast Extract 0.5%

NaCl 1.0%

Agar Powder㻌 1.5 %

䠷Lysis Buffer䠹

50 mM Tris-HCl (pH7.8 at 25

䉝)

2 mM EDTA

2 mM DTT

䠷䝀䝹䜝㐣䜽䝻䝬䝖䜾䝷䝣䜱䞊⏝ Buffer䠹

50 mM Tris-HCl (pH7.8 at 25

䉝)

2 mM EDTA

2 mM DTT

(38)

30 1䠊⳦

⳦య◚○

㞟⳦䛧䛯⳦య䜢

10

ಸ㔞(w/v)䛾

Lysis Buffer

䛻ᠱ⃮䛧䠈1/100 㔜㔞䛾䝸䝌䝏䞊䝮䜒ຍ䛘 ᐊ 䛷

1

᫬㛫ᨩᢾ䛧䛯䚹ḟ䛻ị୰䛻䛚䛔䛶㉸㡢Ἴ◚○ᶵ䛷⳦య◚○䜢⾜䛳䛯䚹㐲ᚰศ 㞳 (14,500 rpm䠈30ศ䠈4 䉝) 䛻䜘䜚ỿẊ≀䜢㝖ཤ䛧䠈ୖΎ⏬ศ䜢ᅇ཰䛧䛯䚹

2䠊㝖

㝖᰾㓟ฎ⌮

⳦య◚○ᚋ䛾ୖΎ⏬ศ䛻ᐊ 䛷ྠయ✚䛾

5 %

䝇䝖䝺䝥䝖䝬䜲䝅䞁⁐ᾮ䜢䜖䛳䛟䜚䛸⁲ୗ䛧䠈

⁲ୗᚋᐊ 䛷

1

᫬㛫ᨩᢾ䛧䛶㝖᰾㓟䜢⾜䛳䛯䚹ᨩᢾᚋ䠈㐲ᚰศ㞳(14,500 rpm䠈30 ศ䠈

4

䉝) 䛻䜘䜚ỿẊ䛧䛯᰾㓟䜢㝖ཤ䛧䠈ୖΎ⏬ศ䜢ᅇ཰䛧䛯䚹

3䠊◲

◲㓟䜰䞁䝰䝙䜴䝮ศ⏬

㝖᰾㓟ᚋ䛾ୖΎ⏬ศ䛻䠈ị୰䛷ᨩᢾ䛧䛺䛜䜙◲㓟䜰䞁䝰䝙䜴䝮䜢⤊⃰ᗘ

55 %㣬࿴䛸䛺䜛

䜎䛷䜖䛳䛟䜚䛸ῧຍ䛧䠈ῧຍᚋ䛥䜙䛻

1

᫬㛫ᨩᢾ䛧䛯䚹᧠ᢾᚋ䠈㐲ᚰศ㞳

(14,500 rpm䠈 30

ศ䠈4 䉝) 䛻䜘䜚ỿẊ⏬ศ䜢ᅇ཰䛧䛯䚹

4䠊䝀

䝀䝹䜝㐣䜽䝻䝬䝖䜾䝷䝣䜱䞊

䛣䛾ỿẊ⏬ศ䜢㐺ᙜ㔞䛾䝀䝹䜝㐣

Buffer 䛻⁐ゎ䛧䠈㐲ᚰศ 㞳 (14,500 rpm䠈10

ศ䠈

4

䉝) 䛻䜘䜚ỿẊ䛧䛯ኚᛶ䝍䞁䝟䜽㉁䜢㝖ཤ䛧䠈ୖΎ⏬ศ䜢ᅇ཰䛧䛯䚹䛭䛾䝃䞁䝥䝹䜢䝣䜱

䠷㏱ᯒ⏝ Buffer䠹

50 mM Tris-HCl (pH7.8 at 25

䉝)

2 mM EDTA

2 mM DTT

䠷㝜䜲䜸䞁஺᥮䜽䝻䝬䝖䜾䝷䝣䜱䞊⏝

Buffer䠹

50 mM Tris-HCl (pH7.8 at 25

䉝)

2 mM EDTA

2 mM DTT

1 M NaCl

(39)

31

䝹䝍䞊 ℐ 㐣䛧䛯 ᚋ 䠈

AKTA-FPLC sysyem

䛻タ ⨨ 䛧䛯䝀䝹䜝㐣䜽䝻䝬䝖䜾䝷䝣䜱䞊

(superdex 200 Increase (volume 25 mL))

䛻䜰䝥䝷䜲䛧䛯䚹䝢䞊䜽⏬ศ䜢

SDS-PAGE

䛷☜ㄆ䛧䠈GroEL䛜⁐ฟ䛧䛯⏬ศ䜢ᅇ཰䛧䛯䚹

5

䠊㝜㝜䜲䜸䞁஺᥮䜽䝻䝬䝖䜾䝷䝣䜱䞊

ᅇ ཰ 䛧䛯 ྛ ⏬ ศ 䜢

AKTA-FPLC system

䛻タ ⨨ 䛧䛯㝜䜲䜸䞁 ஺ ᥮ 䜽䝻䝬䝖䜾䝷䝣䜱䞊

(Resource-Q (volume 6 mL))

䛻䜰䝥䝷䜲䛧䛯䚹

0.1-1 M NaCl

䛾䜾䝷䝆䜶䞁䝖䛷⁐ฟ䛧䛯䚹䝢䞊䜽⏬ศ䜢

SDS-PAGE

䛷☜ㄆ䛧䠈GroEL䛜⁐ฟ䛧䛯⏬ศ䜢ᅇ཰䛧䛯䚹

6䠊㏱

㏱ᯒ䠈⢭〇☜ㄆ

㏱ᯒ⏝

Buffer

䛷୍ᬌ㏱ᯒ䜢⾜䛔䠈㏱ᯒᚋ⺮ⓑᐃ㔞䜢⾜䛳䛯䚹䛭䛾ᚋ䠈SDS-PAGE䛻䜘䜚

⢭〇ᶆရ䛾☜ㄆ䜢⾜䛳䛯䚹

⢭〇䛻䛚䛡䜛ヲ⣽䛺ᡭ㡰䛿ᅗ

2-1

䛻グ䛧䛯䚹

(40)

32

⢭〇䛾ὶ䜜 (GroEL)

ᅗ2-1. GroEL䛾⢭〇㐣⛬

(41)

33 22-2-9

䠊

CD

ᐃ

GroEL WT

ཬ䜃

GroEL G192X

ኚ␗య䛾

2

ḟᵓ㐀䜢ㄪ䜉䜛䛯䜑䛻䠈CD ᐃ䜢⾜䛳䛯䚹

䠷 ᐃ⁐ᾮ⤌ᡂ䠹

50 mM Tris-HCl (pH7.8 at 37

䉝)

10 mM Mg(CH

3

COO)

2

20 mM KCl 2 mM DTT 200 μg/ml GroEL

JASCO J-820 Spectrophotometer

౑⏝䛧䛶䠈෇೫ග஧Ⰽᛶ䠄CD䠅䝇䝨䜽䝖䝹䜢 ᐃ䛧䛯䚹ㄪ〇䛧䛯䝃䞁䝥䝹䛿䠈ග㊰㛗

1 mm

䛾▼ⱥ䝉䝹䜢⏝䛔䠈❅⣲䜺䝇ὶ㏿

2 L/ศ䠈25°C

䛷௨ୗ

䛻♧䛩 ᐃ᮲௳䛷 ᐃ䛧䛯䚹

䠄 ᐃ᮲௳䠅ឤᗘ䠖Low䠄1000 mdeg䠅䠈㛤ጞἼ㛗䠖250 nm䠈⤊஢Ἴ㛗䠖200 nm䠈䝕䞊䝍ྲྀ

㎸㛫㝸䠖0.1 nm䠈㉮ᰝ䝰䞊䝗䠖continuous䠈㉮ᰝ㏿ᗘ䠖50 nm/ศ䠈䝺䝇䝫䞁䝇䠖0.25 sec䠈䝞䞁䝗ᖜ䠖1.0 nm䠈✚⟬ᅇᩘ䠖10ᅇ

2-2-10

䠊

ANS

⺯ග ᐃ

䜰䝙䝸䝜䝘䝣䝍䝺䞁䝇䝹䝩䞁㓟(ANS䠅䛿㟁Ⲵ⛣ືᆺ⺯ගⰍ⣲䛾

1

䛴䛷䠈⣧Ỉ୰䛻ẚ䜉␯Ỉ

⎔ቃୗ䛻⨨䛛䜜䜛䛸䛭䛾⺯ගᙉᗘ䛜ⴭ䛧䛟ቑ኱䛩䜛䛯䜑䠈␯Ỉᛶ䝥䝻䞊䝤䛸䛧䛶ᗈ䛟฼⏝䛥䜜䛶䛝䛯䚹䛣䛾ᛶ㉁䜢฼⏝䛧䠈㔝⏕ᆺ

GroEL

䛸

G192X

ኚ␗య䛾␯Ỉᛶ⎔ቃ䛾㐪䛔䜢ㄪ䜉䛯䚹

(42)

34 ANS

⺯ග䝇䝨䜽䝖䝹 ᐃ䛿䠈JASCO FP-6300 Fluorescence Spectrophotometer䠈ග

㊰㛗

1 cm

䛾▼ⱥ䝉䝹䜢⏝䛔䛶䠈௨ୗ䛾 ᐃ᮲௳䛷⾜䛳䛯䚹

䠄 ᐃ᮲௳䠅 ᐃ䝰䞊䝗䠖Emission䠈ບ㉳䝞䞁䝗ᖜ䠖5 nm䠈⺯ග䝞䞁䝗ᖜ䠖5 nm䠈䝺䝇䝫䞁䝇䠖Medium䠈ឤᗘ䠖Medium䠈ບ㉳Ἴ㛗䠖371 nm䠈㛤ጞἼ㛗䠖400 nm䠈⤊஢Ἴ㛗䠖600 nm䠈

ྲྀ㎸㛫㝸䠖0.5 nm䠈㏿䛥䠖100 nm/ศ䠈✚⟬䠖5ᅇ

22-2-11

㸬ࢤࣝℐ㐣ࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡿศ㞳ศᯒ

GroEL G192X

ྛኚ␗యࡢᅄḟᵓ㐀ࡢኚ໬ࢆศᯒࡍࡿࡓࡵ࡟㸪ࢤࣝℐ㐣ࢡ࣐ࣟࢺࢢ

ࣛࣇ࢕࣮࡟ࡼࡿศᯒࢆ⾜ࡗࡓࠋࢤࣝℐ㐣ศᯒ࡟⏝࠸ࡿ࣒࢝ࣛࡣ㸪 ࢱࣥࣃࢡ㉁ศᏊࡢ኱

ࡁࡉࡈ࡜࡛⁐ฟࡉࢀࡿ㏿ᗘࡀ␗࡞ࡿࠋࡑࡢࡓࡵ㸪⁐ฟࡋࡓࢱࣥࣃࢡ㉁ศᏊࡢ࠾࠾ࡼࡑ ࡢศᏊ㔞ࢆィ ࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࠋ⢭〇ࡋࡓ

GroEL G192X

ྛࢧࣥࣉࣝ࡟⤊⃰ᗘ

ࡀ

40 % (v/v)࡜࡞ࡿࡼ࠺࡟࢔ࢭࢺࣥࢆị୰࡟࠾࠸࡚ࡺࡗࡃࡾ࡜⁲ୗࡋࡓࠋࡑࡢᚋ㸪ị

୰࡛

10

ศ㛫ᨩᢾࡋࡓࠋᨩᢾᚋ㸪

14,500 rpm㸪4Υ㸪30

ศ㛫㐲ᚰศ㞳ࡋ㸪ୖΎࢆᅇ཰

ࡋࡓࠋᅇ཰ࡋࡓୖΎࢆࢤࣝࢁ㐣ࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ศᯒ⏝

Buffer

୍࡛ᬌ㏱ᯒࡋࡓᚋ㸪

Buffer

ࢆ ஺ ᥮ ࡋ 㸪 ࡉ ࡽ ࡟

2

᫬ 㛫㏱ ᯒ ࢆ ⾜ ࡗ ࡓ ࠋ ㏱ ᯒ ᚋ 㸪

Vivaspin 500 (MWCO 10,000(GE Healthcare))࡟ࡼࡾ⃰⦰ࢆ⾜ࡗࡓࠋࡑࡢᚋ 5ȣM

࡜࡞ࡿࡼ࠺࡟

0.5 ml

࡛ㄪ

ᩚࡋࡓࢧࣥࣉࣝࢆணࡵࢤࣝℐ㐣ࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ศᯒ⏝

Buffer

࡛ᖹ⾮໬ࢆ⾜ࡗ࡚

࠸ࡓ

AKTA-FPLC system

䛻タ⨨䛧䛯䝀䝹䜝㐣䜽䝻䝬䝖䜾䝷䝣䜱䞊 (superdex 200 Increase

(volume 25 mL))

䛻䜰䝥䝷䜲䛧䛯䚹⁐ฟ᮲௳ࡣ㸪ὶ㏿

0.75 ml/min

㸪0.5 ml=1 Fraction

࡛⾜࠸㸪UV280nmࡢ⣸እග࡟ࡼࡾ⁐ฟࢧࣥࣉࣝࢆィ ࡋࡓࠋGroELࡢࣆ࣮ࢡࡀࡳࡽ