ᅗ2-9䠊GroEL䛾MDH䠈Rhodanese 䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ ᐃ A䠖MDH refolding assay 㻌 B䠖Rhodanese refolding assay
Spontaneous(䠉)䠖ᇶ㉁䛾⮬Ⓨⓗ䛺⏕䜢♧䛧䛶䛔䜛䚹 C䠖60 ศ䛻䛚䛡䜛MDH,Rhodanese 䛾䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ ᐃ
MDH䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ ᐃ(䕔)䠈Rhodanese 䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ ᐃ(䕔) GroEL G192X䛾್䛿䠈60ศ䛻䛚䛡䜛GroEL WT䛾 ᐃ⤖ᯝ䜢100 %䛸䛧䛶⟬ฟ䛧䛯䚹 ྛ䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ ᐃ䛿3ᅇ ᐃ䛾ᖹᆒ䜢♧䛧䠈3ᅇ ᐃ䛾ᶆ‽ㄗᕪ䜢⟬ฟ䛧䛯䚹
52
ᅗ2-10䠊G192Xኚ␗య䛾䝣䜯䞁䝕䝹䝽䞊䝹䝇య✚䛸䝅䝱䝨䝻䝙䞁ᶵ⬟䛾㛵ಀ 䛭䜜䛮䜜䛾⤖ᯝ䛿60ศⅬ䛷䛾ᩘ್䜢♧䛧䠈MDH䛸Rhodanese 䛾䝸䝣䜷䞊䝹䝕䜱䞁䜾
άᛶ ᐃ䛾⤖ᯝ䛿䠈GroEL WT䛾 ᐃ⤖ᯝ䜢100%䛸䛧䛶⟬ฟ䛧䛯䚹 ATPase assay (GroES㠀Ꮡᅾୗ䠖䖃)䠈ATPase assay (GroESᏑᅾୗ䠖䚽) MDH 䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ ᐃ(䕔)䠈Rhodanese 䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ ᐃ(䕺)
䛭䜜䛮䜜䛾⤖ᯝ䛿䠈3ᅇ ᐃ䛾ᖹᆒ್䜢䝥䝻䝑䝖䛧䠈䜶䝷䞊䝞䞊䛿ᶆ‽ㄗᕪ䜢♧䛩䚹
ᅗ
2-10
䛻䛚䛡䜛GroEL G192X
ྛኚ␗య䛾䝣䜯䞁䝕䝹䝽䞊䝹䝇య✚䛸ATPase
άᛶ䠈MDH䠈Rhodanese
䛾䝸䝣䜷䞊䝹䝕䜱䞁䜾ά ᛶ ᐃ 䛾⤖ ᯝ 䛷䛿䠈GroEL WT
䛸ẚ ㍑ 䛧䛶GroEL G192X
ྛ ኚ ␗ య 䛿᭦ 䛻ά ᛶ 䛜ప ୗ 䛧䛶䛔䜛䛾䛜䜏䜙䜜䛯䚹䛣䛾⤖ ᯝ 䜢ཷ 䛡䛶䠈GroEL㻌 G192X
ྛኚ␗య䛾cis䉄 ring
䛻䛚䛡䜛Open
≧ែ䛻䛺䛳䛶䛔䜛ᗘྜ䛔䜢䜘䜚ᐃ㔞ⓗ䛺ᡭἲ䛷ゎᯒ䛩䜛䛯䜑䛻
QCM
䛷䛾 ᐃ䜢⾜䛖䛣䛸䛻䛧䛯䚹22-3-4䠊GroEL G192X
ኚ␗య䛸GroES
䛾┦స⏝ゎᯒ㐣ཤ䛾◊✲䛻䛚䛔䛶䠈GroEL G192Wኚ␗య䛿
ATP
㠀Ꮡᅾୗ䛷GroES
䛸⤖ྜ䛩䜛䛸䛔 䛖䛣䛸䛜᫂䜙䛛䛸䛺䛳䛶䛔䜛䛯䜑䠈QCM(Qurtz Crystal Microbalance assay)䛻䛚䛔䛶䠈ྛኚ␗య䛸
GroES
䛸䛾ぶᛶ䜢ATP
㠀Ꮡᅾୗ䛷䛹䛾䜘䛖䛺ኚ䛜䜏䜙䜜䜛䛛䛾ᐇ㦂䜢⾜䛳䛯䚹53
ᅗ2-11䠊ATP㠀Ꮡᅾୗ䛻䛚䛡䜛GroEL G192Xኚ␗య䛸GroES䛾┦స⏝ ᐃ 䝩䝇䝖ศᏊ䛸䛧䛶ESC7䜢50䃛M䝉䞁䝃䞊ୖ䛻ᅛᐃ䛧䛶䛔䜛䚹
䝀䝇䝖ศᏊ(ῧຍ䛧䛯GroEL) 䜢20 nM ῧຍ䛧䛶GroEL 䛸GroES䛸䛾┦స⏝䜢ゎᯒ䛧䛯䚹 GroEL WT䛿ATP㠀Ꮡᅾୗ䛷䛿GroES䛸⤖ྜ䛧䛺䛔䛾䛷䠈
ATPᏑᅾୗ(+ATP)䠈㠀Ꮡᅾୗ(䇲ATP)䛻䛚䛡䜛┦స⏝ゎᯒ䛾⤖ᯝ䜢䛭䜜䛮䜜♧䛩䚹
54
ᅗ2-12䠊ATPᏑᅾୗ䠈㠀Ꮡᅾୗ䛻䛚䛡䜛GroEL G192Nኚ␗య䛸GroES䛾┦స⏝
ATP㠀Ꮡᅾୗ䛻䛚䛡䜛GroEL WT 䛸GroES(䖃) ATP㠀Ꮡᅾୗ䛻䛚䛡䜛GroEL G192N䛸GroES(䖃) ATPᏑᅾୗ䛻䛚䛡䜛GroEL WT䛸GroES(䖃) ATPᏑᅾୗ䛻䛚䛡䜛GroEL G192N䛸GroES(䖃)
-200 -150 -100 -50 0
0 50 100 150 200 250 300
Δ 㻲 㻌 (H z)
Time (s)
55
ᅗ
2-11
䛻♧䛧䛯QCM
䛾⤖ᯝ䜘䜚䠈GroEL WT䛿ATP
䛾㠀Ꮡᅾୗ䛷䛿ᇶᯈୖ䛻ᅛᐃ䛧䛯
GroES
䛸䛿⤖ྜ䛧䛺䛔䚹䛧䛛䛧䠈ATP䛾Ꮡᅾୗ䛷䛿ᇶᯈୖ䛻ᅛᐃ䛧䛯GroES
䜈⤖ྜ䛧䛶䛔䜛䚹䛥䜙䛻䠈ATP 䛾㠀Ꮡᅾୗ䛻䛚䛔䛶
GroEL G192X
ྛኚ␗య䛿ATP
Ꮡᅾୗ䛾GroEL WT
䜘䜚䜒䛥䜙䛻GroES
䛾⤖ྜ䛜⏕䛨䛶䛔䜛䚹GroEL䛾Gly192
䜢䛾䜰䝭䝜㓟ṧᇶ 䛻⨨䛧䛯䛣䛸䛷GroEL G192W
䛿ATP
㠀Ꮡᅾୗ䛷䛥䛘䜒cis䉄 ring
ഃ䛻GroES
䛜⤖ྜ䛧䛶䛔䛯䚹䛣䜜䜘䜚䠈䛾
GroEL G192X
ྛኚ␗య䜒䠈䜰䝢䜹䝹䝗䝯䜲䞁䛜Open
≧ែ䛻䛺䛳䛶 䛚䜚䠈cis䉄 ring
ഃ䛻䛿GroES
䛜⤖ྜ䛩䜛䛣䛸䛷ᇶ㉁䛾䝸䝣䜷䞊䝹䝕䜱䞁䜾䜢ຓ䛧䛶䛔䛺䛔 䛣䛸䛜⪃䛘䜙䜜䜛䚹䜎䛯䠈ᅗ2-12
䛷♧䛧䛯䜘䛖䛻䠈ATP Ꮡᅾୗ䛸㠀Ꮡᅾୗ䛷GroEL G192N
ኚ␗య䛿ᇶᯈ䛻ᅛᐃ䛧䛯GroES
䜈䛾⤖ྜ䛻㐪䛔䛜䜏䜙䜜䛯䚹䛣䛾⤖ᯝ䛸GroEL G192X
ኚ␗య㛫䛻䛚䛡䜛䝅䝱䝨䝻䝙䞁άᛶ䛾㐪䛔䛛䜙trans䉄 ring
ഃ䛷䝅䝱䝨䝻䝙䞁ᶵ⬟䛜ാ䛔䛶䛔 䜛ྍ⬟ᛶ䜒⪃៖䛥䜜䜛䚹䛭䛾䛯䜑䠈trans䇲 ring
ഃ䛻䛚䛡䜛GroEL
䛾ᶵ⬟䜢㝖䛧䛯GroEL
䝅䞁䜾䝹䝸䞁䜾ኚ␗యSR-1
ኚ␗య䜢⏝䛔䛯ᶵ⬟ゎᯒ䜢⾜䛳䛯䚹䛣䛾SR-1
䛻䛚䛡䜛Gly192
ṧᇶ䜢Val䠈Ile䠈Trp
䛻䛭䜜䛮䜜⨨䛧䛯ኚ␗య䜢స〇䛧䠈䛣䜜䜙䛾ኚ␗య䜢⏝䛔䛯䝅䝱䝨 䝻䝙䞁άᛶ䜢 ᐃ䛩䜛䛣䛸䛷䠈䜘䜚ヲ⣽䛺GroEL G192X
ኚ␗య䛾䝅䝱䝨䝻䝙䞁ᶵ⬟䜢ゎᯒ䛩 䜛䛣䛸䛻䛧䛯䚹56
ᅗ2-13䠊SR-1 G192X ኚ␗య䛾䝅䝱䝨䝻䝙䞁ᶵ⬟ゎᯒ A䠊SR-1 G192X ኚ␗య䛾ATPຍỈศゎάᛶ
GroES㠀Ꮡᅾୗ(䕔)䠈GroESᏑᅾୗ(䕔) B䠊SR-1 G192Xኚ␗య䛾䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ
MDH (䕔)䠈Rhodanese (䕔)
57
GroEL㻌 G192X
ኚ␗య䛿䠈ATP䛾㠀Ꮡᅾୗ䛷GroES
䛜⤖ྜ䛩䜛䛣䛸䜢♧䛧䛯䚹䝅䝱䝨䝻 䝙䞁άᛶ䛾 ᐃ䛻䛚䛔䛶䠈Gly192ṧᇶ䜢⨨䛧䛯ṧᇶ䛻䜘䛳䛶άᛶ䛻㐪䛔䛜䜏䜙䜜䛯䚹䛣䛾 㐪䛔䛿䠈ᇶ㉁䛜ATP
㠀Ꮡᅾୗ䛷GroES
䛜⤖ྜ䛩䜛cis
䉄ring ഃ䛺䛾䛛䠈䜎䛯䛿ATP
Ꮡᅾୗ䛷
GroES
䛜⤖ྜ䛩䜛䛸⪃䛘䜙䜜䜛trans
䉄ring ഃ䛷䝸䝣䜷䞊䝹䝕䜱䞁䜾䛜⿵ຓ䛥䜜䛯⤖ᯝ 䜢ᫎ䛧䛶䛔䜛䛸⪃䛘䜙䜜䛯䚹䛭䛣䛷䠈䛹䛱䜙䛾䝸䞁䜾䛷䝸䝣䜷䞊䝹䝕䜱䞁䜾䛾ຓ䜢⾜䛖䛛䜢䜘䜚 ヲ⣽䛻ゎᯒ䛩䜛䛯䜑䛻䠈GroEL䛾䝅䞁䜾䝹䝸䞁䜾ኚ␗య(SR-1)䛻䛚䛡䜛Gly192
ṧᇶ䜢䛾 䜰䝭䝜㓟ṧᇶ䛻⨨䛧䛯SR-1 G192X
ኚ␗య䜢స〇䛧䠈䛭䛾䝅䝱䝨䝻䝙䞁ᶵ⬟䛾ゎᯒ䜢⾜䛳 䛯䚹SR-1 G192Xኚ␗య䛷䛒䜜䜀䠈cis -ring
ഃ䛾䜏䛷䛾ᶵ⬟䛜⤖ᯝ䛻ᫎ䛥䜜䜛䛣䛸䛜ᮇᚅ 䛥䜜䜛䚹ᅗ2-13A
䛻GroEL WT䠈SR-1 WT䠈SR-1 G192X
ኚ␗య䛾ATP
ຍỈศゎάᛶ䛾⤖ᯝ䜢♧䛧䛯䚹䛣䛾⤖ᯝ䛻䛚䛔䛶䠈SR-1 WT䛿
GroEL WT 䛾༙ศ⛬ᗘ䛾άᛶ䜢♧䛧䠈SR-1 G䛾༙ศ⛬ᗘ䛾άᛶ䜢♧䛧䠈SR-192X
ኚ␗య䛿SR-1 WT
䜘䜚䜒䛥䜙䛻పୗ䛧䛯⤖ᯝ䜢♧䛧䛶䛔䛯䚹䛣䜜䛿䠈2䛴䛾䝸䞁䜾䛜1
䛴䛻䛺䛳䛯䛣䛸䛷ATP
䛾ຍỈศゎຠ⋡䛜పୗ䛧䛯䛣䛸䛜⪃䛘䜙䜜䜛䚹䛥䜙䛻䠈GroES Ꮡᅾୗ䛷䛿
GroES
㠀Ꮡᅾୗ䜘䜚䜒䛭䜜䛮䜜άᛶ䛜పୗ䛧䛶䛔䛯䛣䛸䛛䜙GroES
䛸䛾┦స⏝䛜⪃䛘䜙䜜䛯䚹ᅗ
2-13B
䛷♧䛧䛯Rhodanese refolding assay
䛾⤖ᯝ䛻䛚䛔䛶䠈SR-1 WT䛿GroEL WT
䛾༙ศ⛬ᗘ䛾䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ䜢♧䛧䛶䛚䜚䠈SR-1 G192Xኚ␗య䛿⨨䛧䛯䜰䝭䝜㓟ṧᇶ䛾䛝䛥䛻䜘䜚άᛶ䛜పୗ䛧䛶䛔䜛䛾䛜䜏䜙䜜䛯䚹䛭䛾୍᪉䛷䠈MDH 䜢ᇶ㉁
䛸䛧䛯䝸䝣䜷䞊䝹䝕䜱䞁䜾άᛶ䛻䛚䛔䛶䠈SR-1 WT䛿䜋䛸䜣䛹άᛶ䜢♧䛧䛶䛔䛺䛛䛳䛯䚹䝅䞁䜾 䝹䝸䞁䜾ኚ␗య
SR-1
䛿䝸䞁䜾㛫䛾䜰䝻䝇䝔䝸䝑䜽䝅䜾䝘䝹䛜⏕䛨䛺䛔䛯䜑䛻୍ᗘ䛰䛡䝅䝱䝨䝻 䝙䞁䝃䜲䜽䝹䛜䜎䜟䜛䛂䝅䞁䜾䝹䝍䞊䞁䜸䞊䝞䞊≧ែ䛃䛻䛺䜛䛸䛥䜜䛶䛔䜛(89)䚹䛣䛾䛯䜑䠈䝅䞁 䜾䝹䝸䞁䜾ኚ␗య䛿MDH
䛜䜻䝱䝡䝔䜱ෆ㒊䛻ⴠ䛸䛧㎸䜎䜜䛯ᚋGroES
䛜ゎ㞳䛫䛪እ䛻ᨺ ฟ䛥䜜䛪άᛶ䛿ᅇ䛧䛺䛔䚹䛧䛛䛧䠈SR-1 G192V䛸SR-1 G192I
ኚ␗య䛿SR-1 WT
䛸ẚ㍑䛧䠈㧗䛔άᛶ䜢♧䛧䛶䛔䛯䚹
ᅗ
2-14
ཬ䜃⾲2-2
䛻SR-1 G192I
䛾ATP
Ꮡᅾୗཪ䛿㠀Ꮡᅾୗ䛻䛚䛡䜛GroES
䛸䛾┦స⏝ゎᯒ䛾⤖ᯝ䜢♧䛧䛯䚹ᅗ
2-14
䛻䛚䛔䛶䠈ATP 㠀Ꮡᅾୗ䜘䜚䜒ATP
Ꮡᅾୗ䛷䛿⃰58
ᗘ䛜ୖ䛜䜛䛻䛴䜜䛶䜘䜚
GroES
䛸䜘䜚┦స⏝䛧䛶䛔䜛䜘䛖䛻䜏䛘䜛䚹䛧䛛䛧䠈䛣䛾䜾䝷䝣䜘䜚┦స⏝䛻䛚䛡䜛ྛ䜹䜲䝛䝔䜱䝑䜽䝟䝷䝯䞊䝍䞊䜢⟬ฟ䛧䛯䛸䛣䜝
ATP
䛾Ꮡᅾୗ䠈㠀Ꮡᅾୗ䛻㛵 䜟䜙䛪ᩘ್䛻䛝䛺㐪䛔䛿䜏䜙䜜䛺䛛䛳䛯䚹䛴䜎䜚䠈ATP 䛾᭷↓䛻㛵䜟䜙䛪GroES
䛸䛾┦స⏝䛿ኚ䛧䛺䛔䛾䜢☜ㄆ䛧䛯䚹⾲
2-2
䛻௨๓SPR(⾲㠃䝥䝷䝈䝰䞁ඹ㬆)䛻䛚䛔䛶 ᐃ䛧䛯
ATP
Ꮡᅾୗ䛾GroEL WT
ཪ䛿ATP
Ꮡᅾୗ䠈㠀Ꮡᅾୗ䛻䛚䛡䜛GroEL G192W
ኚ␗య䛸GroES
䛸䛾䜹䜲䝛䝔䜱䝑䜽䝟䝷䝯䞊䝍䞊䜢♧䛧䛯䚹䛣䛾GroEL
䛾ᩘ್䛸SR-1 G192I
ኚ␗య䛸 䛾ᩘ್䜢ẚ㍑䛩䜛䛸䠈SR-1 G192Iኚ␗య䛿GroEL
䜘䜚䜒⤖ྜ㏿ᗘk
on䛾ᩘ್䛜ᑠ䛥䛔䛣䛸 䛛䜙GroES
䛸䛾⤖ྜ䛜㐜䛔䚹䛥䜙䛻䠈SR-1 G192Iኚ␗య䛾ゎ㞳㏿ᗘk
off䛿GroEL WT
䜘 䜚䜒ᑠ䛥䛔䛣䛸䛛䜙GroES
䛸䛾ゎ㞳䛿㐜䛔䚹䛧䛛䛧䠈GroEL G192W ኚ␗య䜘䜚䛿䛝䛔䛣䛸 䛛䜙䠈GroES 䛸䛾 ゎ 㞳 䛜 ᪩ 䛔 䛣䛸 䛜 ☜ ㄆ 䛷 䛝 䛯 䚹 䛣䛾 ⤖ ᯝ 䜘 䜚 䠈SR-1 G192X
ኚ ␗ య 䛿GroEL G192X
䜘䜚䜒GroES
䛜⤖ྜ䛧㞴䛟䠈ゎ㞳䛧᫆䛔ᛶ㉁䜢䜒䛳䛶䛔䜛䛣䛸䛜⪃䛘䜙䜜䜛䚹䛣 䜜䛜๓㏙䛾䝅䝱䝨䝻䝙䞁ᶵ⬟ゎᯒ䛾⤖ᯝ䛻ᫎ䛥䜜䛶䛔䜛䛸⪃䛘䜙䜜䜛䚹59
ᅗ2-14䠊QCM䛻䜘䜛SR-1 G192I䛾ATPᏑᅾୗཪ䛿㠀Ꮡᅾୗ䛷䛾GroES䛸䛾┦స⏝
A䠊ATP㠀Ꮡᅾୗ䛻䛚䛡䜛SR-1 G192I䛸GroES䛸䛾┦స⏝ゎᯒ B䠊ATPᏑᅾୗ䛻䛚䛡䜛SR-1 G192I䛸GroES䛸䛾┦స⏝ゎᯒ
A䠈B䛻䛚䛔䛶䠈5 nM(䖃)䠈10 nM(䖃)䠈15 nM(䖃)䠈20 nM(䖃)䠈25 nM(䖃)䜢♧䛧䛶䛔䜛䚹
⾲2-2䠊ᅗ2-14䜘䜚⟬ฟ䛧䛯GroES䛸SR-1 G192I䛾䜹䜲䝛䝔䜱䝑䜽䝟䝷䝯䞊䝍䞊 Kd(M)䠖ゎ㞳ᐃᩘ䠈kon (M-1䞉S-1)䠖⤖ྜ㏿ᗘ䠈koff(S-1)䠖ゎ㞳㏿ᗘ
*Biacore2000䜢⏝䛧䛯SPR䝇䝨䜽䝖䝹 ᐃ⤖ᯝ(19)䚹