Further Study on the Spon taneous Fold ing of Ch icken Apocytochrom e c

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1 V o l 15,N o 1 Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy Feb c 3 3 (, ) c V 92A 17, V 92A gc17s W gc17s IA EDAN S A EDAN S2Cys214 A EDAN S T rp 259 V 92A c N A po c, V 92A,, c N c,,a EDAN S, Further Study on the Spon taneous Fold ing of Ch icken Apocytochrom e c HAN Xuehai, Y I P ing, TON G Junchao, YAN G Fuyu (N ational L aboratory of B iom acrom olecules, Institute of B iop hy sics, A cadem ia S inica, B eij ing ) Abstract It has been repo rted that ch icken heart apocytoch rom e c has been show n to po ssess a m uch stronger tendency to fo ld spon taneou sly in aqueou s so lu tion than the equ ivalen t from o ther species and V 92A cou ld alter the fo lding p ropen sity of ch icken apocytoch rom e c and its in teraction w ith pho spho lip id In o rder to fu rther study the confo rm ation difference betw een w ild type of ch icken heart apocytoch rom e c and its V 92A m u tan t, tw o m u tan ts,w gc17s and V 92A gc17s, in w h ich the 17 cysteine residue w as rep laced by serine w ere ob tained and exp ressed in E coli By u sing fluo rescence p robe IA EDAN S (reacted on the SH group of 14 cystein residue) the confo rm a2 tion and that of in tram o lecu lar in teraction s betw een W gc17s and V 92A gc17s w ere investigated by A EDAN S fluo rescence em ission spectra, fluo rescence lifetim e and fluo rescence energy tran sfer efficiency O b tained resu lts show ed that com paring w ith the w ild type of ch icken heart apocy2 toch rom e c the V 92A m u tan t appeared less fo lded state bo th in the p resence and ab sence of 510 m o lgl N ac lo 4 It also seem ed that the N term inal of the ch icken heart apocytoch rom e c m igh t no t be invo lved du ring its spon taneou s fo lding Key words A pocytoch rom e c, Fo lding,a EDAN S label, F luo rescence energy tran sfer efficiency c ( c 59, [ 1 A po c) ], A po c A po c [ 2 ], 92 (V 92A ), 3 ( ) (KZ9512B12609) 3 3 :,, ,, V 92A, 92 V al A po c [ 3 ] A po c A po c V 92A T el: (010) , Fax: (010) E2m ail: xhhan@ sun5 ibp ac cn : P IPES, 1, 42P iperazine2bis2( ethanesulfonic acid ) ; 1, 52IA EDAN S, N 2iodoacetyl2N 2( 52sulfo212naph2 thy1) ethylene2diam ine; D TNB, 5, 5 dithiobis ( 22nitroben2 zo ic acid) ; T FE, 2, 2, 22trifluo roethano l, A po : , : China Academic Journal Electronic Publishing House All rights reserved

2 1 : c 93, IA EDAN S 337 nm, m o l - 1 L cm - 1 [ 6 ] Go to A po c, IA EDAN S 14 17,A EDAN S 337 nm, [ 4 ] 14 17, nm,, 280 nm, 300 A po c V 92A 17 Cys 450 nm, A po c 5 Λm o lgl Ser, W gc17s V 92A gc17s, 280 nm,, A po c Cys nm A po c T rp,, Edinbu rgh 299T A po c N,, 280 nm,, 345 nm, 100 n s, A po c, 17 Λm o lgl JM 109 BM H m u ts BL 21 E, pet 23 d, (ΣDA ) (ΣD ) M 13K07 P rom ega; N co g, E = 1- F DA gf D E = 1- ΣDA gσd R B am H g E cor g DN ase P rom ega, R 0, P IPES (T FE ) 1, 52 50%, R nm [ 8 ] IA EDAN S Sigm a, D TNB F luka, D T T Serva, CM 2sephero se CL 6B Phar2 m acia P IPES mm o lgd T T P IPES, mm o lgl P IPES, ph 710, 50 mm o lgl N ac l Λm o lgl, CD, nm T FE [ 2 ], C17S,,, 5 2GTCCA GAAA T GT TCCCA G TCCCA TA CG23 DNA, [ 2 ] Jasco J2720, E llm an D TNB, R iddles nm, 100 nm g [ 5 ] m ggm l A po c 4 m o lg L F24010 T rp Fo β rster [ 7 ], R = R 0 (E (D E3) A po c pstcn T 7 (F DA ) (F D ) P IPES, Apo c 17 IA EDAN S 2 h, P IPES A po c 17 Cys 4 9 h, 2 h [ 2 ], DNA IA EDAN S, A po c DNA 17-20, 4 m o lgl TCC, C17S IA EDAN S T GC, China Academic Journal Electronic Publishing House All rights reserved 1) 1g m ggm l 8 m o lgl 0 99% (T FE), [3 ] A po c 10 Λm o lgl, 1 mm, m in, 1 nm, 4

3 94 15, ( ) V 92A Α,, C17S V 92A 95% C17S, D TNB C17S A po c, A po c V 92A T ab le 1, A po c V 92A 2,W gc17s V 92A gc17s, 17 Table 1 T he th io l content of apocytoch rom e c and its m utants determ ined by titration w ith D TNB P ro tein W ild V 92A W gc17s V 92A gc17s SH ] [p ro tein ]gmo l mo l A EDAN S C17S,, 8 m o lgl, P IPES, CD F ig11, V 92A V 92A gc17s CD, ; W gc17s CD, nm V 92A V 92A gc17s, 200 nm V 92A V 92A gc17s, F ig 1 CD spectra of A EDAN S labeled w ild type ch icken W gc17s (g ) apocytoch rom e c and its m utants V 92A gc17s ( ) after renaturation by dialysis, the open sym bo ls (( are unlabeled samp les W and V 92A, C17S A po c V 92A 213 IAEDANS T FE W g A po c 14 C17S V 92A gc17s Α, IA EDAN S A EDAN S A po c T FE CD Α 208 nm 222 nm A EDAN S 337 nm 276 nm, 222 nm T FE A EDAN S W gc17s V 92A gc17s T FE F ig 1 1 2, V 92A T FE A EDAN S W gc17s V 92A g T ab le 2 CD C17S T FE V 92A N ac lo 4 P IPES, W gc17s V 92A g, 40% 60% T FE C17S, T FE,W gc17s N 14 A EDAN S nm V 92A gc17s, nm, N, CD [ 3 ] V 92A T FE, W gc17s C17S A EDAN S V 92A gc17s, China Academic Journal Electronic Publishing House All rights reserved )

4 1 : c 95 Table 2 Comparison of intrinsic fluo rescence intensity, w avelength, life tim e and energy transfer efficiency betw een W gc17s and V 92A gc17s 0 mo lgl N ac lo 4 5 mo lgl N ac lo 4 W gc17s (n= 5) V 92A gc17s (n= 5) W avelengthgnm F luo rescence intensity L ife tim egns FET distance 3 gnm W avelengthgnm F luo rescence intensity L ife tim egns FET distance 3 gnm The fluo rescence energy transfer distance is the average distance betw een T rp259 and A EDAN S group s attached at Cys214 fo r the two m utants of chicken apocytochrom e c, the average distances cal2 culated by tryp tophan fluo rescence intensity changes from F ig 3 F ig 2 T FE titration curves fo r A EDAN S labeled w ild type chicken apocytochrom e c W gc17s ( g ) and its m utants V 92A gc17s ( ) after renaturation by dialysis, the open sym bo ls (( ) are unlabeled samp les W and V 92A, fitting curves are derived from the TFE binding model[ 3 ] N Fo β rster T rp N ac lo 4,W gc17s V 92A g A EDAN S T ab le 2 F ig13 C17S, 5 m o lgl N ac lo 4 W gc17s [ 9, Α ], V 92A gc17s N ac lo 4 14 A po c N, Α,, nm 340 nm,, A EDAN S Α, A po c, A EDAN S N ac lo 4, N, 5 m o lgl N ac lo 4 W gc17s V 92A gc17s N ac lo 4,, W gc17s 0 m o lgl N ac lo 4, V 92A gc17s,,,,,,w gc17s V 92A gc17s,, 59, 14, V 92A gc17s, 5 W gc17s V 92A gc17s m o lgl N ac lo 4 W gc17s V 92A gc17s, W gc17s, A EDAN S A EDAN S T rp N ac lo IA EDAN S 337 nm,, W gc17s V 92A gc17s F ig13a B N ac lo 4 5 m o lgl N ac lo 4 A EDAN S2W gc17s A EDAN S2V 92A gc17s,, V 92A China Academic Journal Electronic Publishing House All rights reserved

5 96 15 F ig 3 F luo rescence energy transfer curves of A EDAN S la2 beled W gc17s ( ) and V 92A gc17s (222) in the ab2 sence o r p resence of N ac lo 4 ( unlabeled W gc17s, unlabeled V 92A gc17s) (A ) 0 mo lgl N ac lo 4; (B) 5 0 mo lgl N ac lo 4, V 92A gc17s W gc17s Cys214 T rp 259 V 92A gc17s V 92A 4 m o lgl, A EDAN S A po c T rp T rp W gc17s V 92A gc17s T rp 259 A EDAN S2Cys214, T ab le A po c, V 92A,, A po c,, 311 C17S Apo c A po c 92 V aline,, C17S A po c F ig12 T FE C17S A po c V 92A, A po c N 312 V92A Apo c P IPES, W gc17s V 92A gc17s, Cys214 T rp nm,, A po c Go to A po c [ 4 ], N ac lo 4 A po c, V 92A gc17s Cys214 T rp259 W gc17s V 92A gc17s 5 m o lgl N ac lo 4 A po c,, V 92A C A po c A EDAN S, N, A po c 280 nm, W gc17s V 92A gc17s F ig13a ( ), CD China Academic Journal Electronic Publishing House All rights reserved

6 1 : c V al,,, A la A po c N 1 15, 5 m o lgl N ac lo 4 V 92A gc17s W gc17s, V 92A A po c [ 10, V 92A ], C, V 92A C, A po c C V 92A gc17s 28 39,, D Sho rtle ( ) F lanagan SN ase [ 9 ], V 92A m +, 1, 313 N Apo c A po c, 14 A EDAN S A po c N, A EDAN S, : N 1 15 A po c ph 4 H am ada D, Ho sh ino M, Kataoda M, F ink A L, Go to Y Interm e2, diate confo rm ational states of apocytochrom e c B iochem istry, 2 : (1) W g C17S V 92A gc17s N A EDAN S, ; (2) c 3 nm, A po c, Cyṫ c, 59 A po c, 14,, T rp A EDAN S nm,, A po c, References,,, c B (W ang X ianshu, Tong Junchao, H an Xuehai, Yang Fuyu Co rrelation betw een unfo lded states of apocytoch rom e c and its ability to pass lip id bilayer S cience in Ch ina, Series B) 1994, 24: Tong J C, Zhu L Q, Yang F Y C loning and h igh2level exp res2 sion of chicken apocytochrom e c gene in E scherichia coli B iochem M ol B iol Int, 1995, 36: Tong J C, Zhu L Q, Yang F Y V 92A m utation aitered the fo ld2 ing p ropenstiy of chicken apocytochrom e c and its interaction w ith pho spho lip ids B iochem istry U SA, 1996, 35: U SA, 1993, 32: R iddles P W, B lakeley R L, Zerner B Reassessm ent of ellm an s reagenṫ M ethod s in E nzym ology, 1983, 91: H udson E N, and W eber G Synthesis and characterization of two fluo rescent sulfhydryl reagents B iochem istry, 1973, 12: 7 Fo β rster T H In: Sinanoglu O ed M od ern Q uantum Chem istry N ew Yo rk: A cadem ic P ress, 1965: Go to Y, Calciano L J, F ink A L A cid2induced fo lding of p ro2 teins P roc N atl A cad S ci U SA, 1990, 87: F lanagan J M, Kataoka M, Fujisaw a T, Engelm an D M M uta2 tions can cause large changes in the confo rm ation of a denatured p ro tein B iochem istry U SA, 1993, 32: , 1 c (T ab le 2), A po c ( (H ang Xuehai, Yang Fuyu Further study on the spontaneous partial fo lding of chicken heart apocytochrom e c ), Ch in B iochem J ), 1996, 5: China Academic Journal Electronic Publishing House All rights reserved

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