Stability and Unfolding Mechanism of the N-terminal -Hair pin from [2Fe-2S] Ferredoxin I by Molecular Dy nam ics Sim u la tions

Jour nal of the Chi nese Chem i cal So ci ety, 2003, 50, 799-808 799 Stability and Unfolding Mechanism of the N-terminal -Hair pin from [2Fe-2S] Ferredoxin I by Molecular Dy nam ics Sim u la tions Hsuan-Liang Liu* ( ) and Yuan-Ming Lin ( ) De part ment of Chem i cal En gi neering, Na tional Tai pei Uni ver sity of Tech nol ogy, Tai pei 106, Tai wan, R.O.C. The sta bil ity and un fold ing mech a nism of the N-terminal -hair pin of the [2Fe-2S] ferredoxin I from the blue-green alga Aphanothece sa crum in pure meth a nol, 40% (v/v) meth a nol-water, and pure wa ter sys tems were in ves ti gated by 10 ns mo lec u lar dy nam ics sim u la tions un der pe ri odic bound ary con di tions. The -hair - pin was mostly in its na tive-like state in pure meth a nol, whereas it un folds dra mat i cally fol low ing the zip-up mech a nism when it was placed in pure wa ter. Both interstrand and in side-turn hy dro gen bonds ac count for the sta bil ity of the -hair pin in its na tive-like con for ma tion, whereas hy dro pho bic in ter ac tions among nonpolar side chains are re spon si ble for main tain ing its sta ble loop-like in ter me di ate struc tures in 40% (v/v) meth a - nol- water. Re ducing sol vent po lar ity seems to in crease the sta bil ity of the -hair pin in its na tive-like struc ture. Meth a nol is likely to mimic the par tially hy dro pho bic en vi ron ment around the N-terminal -hair pin by the sub se quent -he lix. Key words: Blue-green alga Aphanothece sa crum; Meth a nol; Pe ri odic bound ary con di tions; Zip-up mech a nism; Hy dro gen bonds; Hy dro pho bic in ter ac tions; Po lar ity. IN TRO DUC TION As the knowl edge of var i ous DNA and pro tein se quences from dif fer ent or gan isms rap idly in creases, proteomics has be come ex tremely im por tant in the post-genomic era. The tasks of proteomics can be cat e go rized into three main ac tiv i - ties: 1) iden ti fy ing all the pro teins made in a given cell, tis sue or or gan ism; 2) de ter min ing how those pro teins join forces to form com pli cated net works; and 3) out lin ing the pre cise three-dimensional struc tures of the tar get pro teins for fur ther ra tio nal drug de sign. Both x-ray crys tal log ra phy and NMR spec tros copy have been widely used for the struc tural de ter - mi na tion of pro teins. 1 In ad di tion, much at ten tion has been paid to in ves ti gate the fold ing/un fold ing mech a nisms of pro - teins un der dif fer ent con di tions. Ever since the early ex per i - ments, 2 pro tein fold ing has been rec og nized to be a spon ta ne - ous event and all the in for ma tion re quired for cor rect fold ing is be lieved to be con tained in the amino acid se quence. Dif - fer ent fold ing mod els (e.g., nu cle ation, early sec ond ary struc - ture for ma tion, or hy dro pho bic clus ter for ma tion) have been pro posed so far to ex plain the com pli cated fold ing mech a - nisms. 3-8 How ever, ac cord ing to the Levinthal par a dox, 9-11 pro tein fold ing can not oc cur by a ran dom search of all pos si - ble con for ma tions. Since nu mer ous ad vances by suc cess ful de sign of cer - tain model pep tides to un der stand pro tein struc ture and fold - ing mech a nisms have been achieved in the past few years, 12-16 the study of small pep tides and pro tein frag ments leads to a better un der stand ing of pro tein fold ing in the early stage, where both -turns and -hair pins have been con sid ered to act as the fold ing ini ti a tion sites. 12,17-26 Ex am ples were given by the in ves ti ga tion of the early events of pro tein fold ing with iso lated pep tide frag ments un der equi lib rium con di tions. 27-29 Pre vi ous stud ies with frag ments from pro tein-gb1 do main (GB1) 12,13,30 and ubiquitin 17,31 pre sented the au ton o mous fold ing of short pep tide mol e cules into -hair pins with var i - ous sta bil ity in aque ous and mixed aque ous-organic sol vent sys tems. More over, it has been in di cated by com puter mo lec - u lar sim u la tions that the time for -turn and -hair pin pep - tides to fold into their na tive state is within ns 32-34 and s, 35 re - spec tively. These prom is ing re sults have made it fea si ble to con duct the pro tein fold ing/un fold ing pro cesses in Silico. The sta bil ity of the -hair pin in aque ous so lu tion has been at trib uted to many fac tors. For ex am ple, al co hols have been shown to oc ca sion ally sta bi lize the struc ture of -hair - pin pep tides due to the rel a tively lo cal hy dro gen bond in ter - ac tions. 13,36 Lo cal hy dro gen bonds seem to play an im por tant role for sec ond ary struc ture sta bi li za tion in sol vents of rel a - tively low po lar ity, whereas the hy dro pho bic in ter ac tions show less in flu ence on sta bi liz ing a pro tein. The ef fect of al - * Cor re sponding au thor. Tel: +886-2-2771-2171 ext. 2542; fax: +886-2-2731711; e-mail: f10894@ntut.edu.tw

800 J. Chin. Chem. Soc., Vol. 50, No. 4, 2003 Liu and Lin co hols can also be ex plained to some ex tent by de creased sol - vent po lar ity. 37-40 In ad di tion to the im por tance of sol vent prop er ties, in trin sic res i due pref er ences and interstrand in ter - ac tions have also been showed not only to pro mote -hair pin for ma tion from var i ous lin ear pep tides, 20,22,41,42 but also to con trib ute to the -hair pin sta bil ity sig nif i cantly. 43-45 Fur - ther more, the -turn res i dues can also be cru cial to the sta bi li - za tion of -hair pin struc ture. 46-48 Both type I and type II -turns are the most abun dant turn mo tifs found in -hair pins so far, 49 prob a bly due to that these turns ex hibit a fa vor able ge om e try to match the right-handed twist of the anti-parallel -sheet. 50 Pre vi ous study has shown that the fold ing/un fold ing of a 19-residue pep tide, cor re spond ing to the first -hair pin of the -am y lase in hib i tor tendamistat, fol lows the so-called zip-up mech a nism (Fig. 1), in which the -turn is formed first fol lowed by hy dro gen bond for ma tion clos ing the hair - pin, and sub se quent sta bi li za tion by side-chain hy dro pho bic in ter ac tions. 25 In ad di tion, Maynard et al. 51 have sug gested that the hy dro pho bic ef fect is the dom i nant sta bi liz ing in ter - ac tion for -hair pin in wa ter by struc tural and ther mo dy - namic anal y ses of the model pep tide. In the pres ent study, we ex am ined the sol vent ef fects on the sta bil ity and the un fold - ing mech a nism of the N-terminal -hair pin of the [2Fe-2S] ferredoxin I from the blue-green alga Aphanothece sa crum by mo lec u lar dy nam ics sim u la tions in dif fer ent aque ousorganic so lu tions. The struc ture of the [2Fe-2S] ferredoxin I from the uni - cel lu lar blue-green alga A. sa crum has been de ter mined by x-ray crys tal log ra phy. 52 It is com posed of four iden ti cal sub - units; each of them con sists of 96 amino ac ids and has a rel a - tive mo lec u lar weight of 10.48 kda (Fig. 2). The amino acid se quence of this pro tein was found to dif fer from that of the [2Fe-2S] ferredoxin from the fil a men tous blue-green alga Spirulina platensis 53,54 by only 30%, 52 lead ing to the ob ser va - Fig. 1. The pos si ble fold ing/un fold ing mech a nisms of -hair pin of Tendamistat pro posed by Bonvin et al. 25 The Zip-up mech a nism shows that the -turn is formed first, fol lowed by hy dro gen bond for ma tion clos ing the -hair pin. In con - trast, the interstrand hy dro gen bonds are formed first, fol lowed by form ing a sta ble -turn struc - ture for the Zip-down mech a nism. Fig. 2. The rib bon struc ture of one of the four sub units of [2Fe-2S] ferredoxin I. 52 The N- and C- ter - minus are in di cated as N and C, re spec tively. The N-terminal -hair pin and the sub se quent -he lix are shown in dark and light blues, re - spec tively. Fig. 3. The ste reo view of the N-terminal -hair pin of [2Fe-2S] ferredoxin I.

Stability and Unfolding Mechanism of -Hairpin J. Chin. Chem. Soc., Vol. 50, No. 4, 2003 tion that both enzymes share a -barrel structure and adopt a common / -roll fold, consisting of four -strands and one -he lix. This fold is sim i lar to the struc ture mo tif in both ubiquitin and GB1. 52,55-57 The four -strands in [2Fe-2S] ferredoxin I from the blue-green alga A. sacrum are formed by the residues Tyr2-Thr9, Gly12-Val20, Lys50-Val52 and Asp84-Glu88 and the -he lix is formed by the res i dues Tyr-23-Glu31. 52 Both ubiquitin and [2Fe-2S] ferredoxin I adopt a hairpin-turn-helix motif where the two -strands near the N-terminus form a stable -hairpin structure connected to the subsequent -helix, which forms a large proportion of the hy dro pho bic core of the pro teins.52 This hair pin-turn-helix motif has become a good model system to investigate the stabil ity of the -hair pin struc ture in a hy dro pho bic en vi ronment.18 RESULTS AND DISCUSSION In the pres ent study, the 20-residue pep tide, ASYKVTLKTPDGDNVITVPD, cor re spond ing to the Nterminal -hair pin from res i dues 1 to 20 of the [2Fe-2S] ferredoxin I from the unicellular blue-green alga A. sacrum taken from the x-ray crystallographic structure (PDB entry F1XI)52 was subjected to 10 ns MD simulations in different solvent systems. To investigate the influence of different solvents on the stability of the initial structure, we submitted the target -hairpin fragment, which was first energy minimized by steepest descent method for 5,000 iterations, into the pure meth a nol, 40% (v/v) meth a nol-water, and pure wa ter systems, respectively. Fig. 4 shows that the starting structure of -hairpin in pure methanol is similar to the crystallographic structure, whereas it significantly deviates from the crystallographic structure when it was placed in the pure water system. The RMSDs of the backbone C of the starting structure in the pure meth a nol, 40% (v/v) meth a nol-water, and pure water systems with reference to the crystallographic structure are 0.6, 2.1, and 2.0 Å, respectively (Fig. 5). These results in- Fig. 5. The RMSDs of the backbone C of the -hairpin during the 10 ns MD simulations in (a) the pure methanol system; (b) the 40% (v/v) methanol-water sys tem; and (c) the pure wa ter system. The horizontal dashed lines in (b) and (c) in di cate the point above which the -hair pin started to unfold. Fig. 4. The energy minimized starting structures of the -hairpin (dark lines) superimposed on the crystallographic structure (light lines) in (a) the pure methanol system; (b) the 40% (v/v) methanol-water system; and (c) the pure water system. Energy minimization was carried out as described in the text. 801

802 J. Chin. Chem. Soc., Vol. 50, No. 4, 2003 Liu and Lin di cate that sol vent does play an im por tant role for sec ond ary struc ture sta bi li za tion, which is con sis tent with the pre vi ous CD and NMR spec tros copy re sults show ing that ad di tion of meth a nol or TFE to an aque ous so lu tion of this -hair pin frag ment pro motes the for ma tion of na tive-like monomeric -hair pin struc ture. 18 The ef fect of al co hols on stim u lat ing sec ond ary struc ture for ma tion has been mainly at trib uted to a strength en ing of intramolecular hy dro gen bond ing. 58 The lower di elec tric con stant in a meth a nol sys tem seems to re sult in less ef fec tive sol va tion of am ide di poles than pure wa ter, lead ing to the sta bi li za tion of the folded state of -hair pin struc ture where intramolecular am ide hy dro gen bonds are en - er get i cally more fa vor able than am ide-solvent intermolec - ular hy dro gen bonds. 18 As can be seen from the crys tal lo graphic struc ture, the N-terminal -hair pin is sub se quently con nected by an -he - lix. It is be lieved that ad di tional in ter ac tions of the -hair pin with the -he lix and other por tions of the polypeptide chain as pre req ui sites for sec ond ary struc ture sta bi li za tion are nec - es sary. 18 The ob ser va tion that the -hair pin struc ture re sem - bles its crys tal lo graphic struc ture in the pure meth a nol sys - tem, whereas it shows a dra matic de vi a tion from its na tivelike struc ture in the 40% (v/v) meth a nol-water and pure wa ter sys tems, strongly in di cates that the less po lar sol vent sys tem is likely to mimic the hy dro pho bic en vi ron ment sur round ing the N-terminal -hair pin pro vided by the sub se quent -he lix, lead ing to sta bi liz ing the -hair pin struc ture. Meth a nol sta bi - lizes the -hair pin struc ture mainly by strength en ing am ideamide hy dro gen bonds rel a tive to am ide-solvent in ter ac tions. As can be seen from Fig. 6(a), the to tal num ber of intra - molecular hy dro gen bonds var ied be tween 5 and 11 dur ing the 10 ns MD sim u la tions in the pure meth a nol sys tem. Here, we de fine the interstrand hy dro gen bonds as the hy dro gen bonds formed be tween the am ide and car bonyl groups of res i - dues Tyr3 and Val18, Val5 and Ile16, and Leu7 and Asn14. The in side-turn hy dro gen bonds are de fined as the hy dro gen bonds formed be tween the am ide and car bonyl groups of res i - dues Thr9 and Gly12 (Ta ble 1). The interside-chain hy dro gen bonds are de fined as the hy dro gen bonds formed be tween charged or po lar side chains lo cated on the same face of the -hair pin. Fig. 6(a) shows that the num ber of the in side- turn hy dro gen bonds re mained at the level of 2 dur ing most of the MD sim u la tion course, while the num ber of the interstrand hy dro gen bonds var ied be tween 3 and 6. It in di cates that hy - dro gen bonds close to the -turn re gion are more sta ble than those lo cated far away from the -turn. Pro10 lo cated in the -turn is likely to re duce the de gree of free dom of the back - bone near the -turn re gion due to the conforma tional re stric - tion ef fect by its cy cli cally bonded struc ture, lead ing to sta bi - (a) (b) (c) 12 10 6 5 4 3 2 1 0 8 6 4 2 0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 1 2 3 4 5 6 7 8 9 10 time (ns) 0 1 2 3 4 5 6 7 8 9 10 time (ns) 0 1 2 3 4 5 6 7 8 9 10 time (ns) Fig. 6. The num ber of hy dro gen bonds formed in the 10 ns MD sim u la tions in (a) the pure meth a nol sys tem; (b) the 40% (v/v) meth a nol-water sys - tem; and (c) the pure wa ter sys tem. @: the to tal num ber of hy dro gen bonds; : the num ber of the interstrand hy dro gen bonds; : the num ber of the in side-turn hy dro gen bonds; : the num ber of interside-chain hy dro gen bonds in the two -strands; : the num ber of interside-chain hy - dro gen bonds in the -turn re gion.

Sta bil ity and Un folding Mech a nism of -Hair pin J. Chin. Chem. Soc., Vol. 50, No. 4, 2003 803 Table 1. The Positions and Lengths of the Hydrogen Bonds in the Initial Crystallographic Structure of the -Hairpin of the [2Fe-2S] Ferredoxin I from the Blue-Green alga A. sacrum 52 C=O group N-H group H-bond classifications H-bond distance (Å) Val18 Tyr03 interstrand 2.89 Tyr03 Val18 interstrand 2.95 Ile16 Val05 interstrand 2.74 Val05 Ile16 interstrand 2.84 Asn14 Leu07 interstrand 2.76 Leu07 Asn14 interstrand 2.89 Gly12 Thr09 inside-turn 2.73 Thr09 Gly12 inside-turn 2.74 liz ing the hy dro gen bonds formed near this re gion. The interside-chain hy dro gen bonds do not con trib ute to sta bi lize the -hair pin struc ture since only 1 or 2 were found dur ing the en tire MD sim u la tion course. Al though the pos si bil ity to solve the pro tein fold ing prob lem by di rect in silico sim u la tions on the atomic scale has not yet be come a re al ity due to the rel a tively small timespan, the in creased com puter power has started to make some of the rel e vant prob lems in pro tein fold ing ame na ble to sim u - la tion meth ods such as mo lec u lar dy nam ics (MD) and Monte Carlo (MC) cal cu la tions. While the lat ter method has been pri mar ily ap plied in the con text of ex plor ing sim pli fied lat - tice or grid mod els of pro tein struc ture, the for mer one has been widely em ployed to gain in sight into the pro tein fold ing pro cess. 59,60 Pro tein fold ing usu ally oc curs on the sec ond time- scale, which has made com puter sim u la tions dif fi cult to reach the re al is tic sit u a tions. To over come this prob lem, the so- called un fold ing sim u la tions have started to at tract in ten - sive in ves ti ga tion since the early 90s. 61-63 The com mon strat - e gies to in duce the un fold ing of a pro tein in its na tive-like struc ture are the tem per a ture jump tech nique, 35,64-68 pres - sure- induced de na tur ation, 69 and chang ing the sol vent. 70 How ever, ther mal de na tur ation is not nec es sar ily sim i lar to pro tein fold ing at phys i o log i cal or room tem per a tures with re spect to pos si ble fold ing/un fold ing path ways, in ter me di - ates sam pled, and tran si tion states. There fore, pro tein fold - ing/un fold ing in duced by dif fer ent sol vents has pro vided an al ter na tive ap proach to in ves ti gate the pos si ble pro tein fold - ing/un fold ing mech a nisms ei ther by in vitro ex per i ments or by in silico sim u la tions. Daura et al. 71 had shown that a short -heptapeptide can fold into its na tive 3 1-he li cal struc ture spon ta ne ously in meth a nol on a 10 ns time-scale sim u la tion. This prom is ing re sult en cour ages us to in ves ti gate the pos si - ble un fold ing mech a nism of the tar get -hair pin in duced by sol vent ef fects with MD sim u la tions. Fig. 7 shows the snap shots of the -hair pin struc ture su - per im posed on the crys tal lo graphic struc ture from a 10 ns MD sim u la tion in the pure meth a nol sys tem. As can be seen, the tar get -hair pin main tains in a sta ble con for ma tion that is closely sim i lar to its na tive-like struc ture dur ing the en tire course of the 10 ns MD sim u la tion. The two -strands of the -hair pin re mained con nected by interstrand hy dro gen bonds, Fig. 7. The snap shots of the -hair pin (blue lines) at (a) 0; (b) 2; (c) 4; (d) 6; (e) 8; (f) 10 ns su per im posed on the crys tal lo - graphic struc ture (black lines) in the pure meth a nol sys tem dur ing the 10 ns MD sim u la tions.

804 J. Chin. Chem. Soc., Vol. 50, No. 4, 2003 Liu and Lin whereas the -turn re gion de vi ated from its rel e vant po si tion in the crys tal lo graphic struc ture af ter 2 ns of sim u la tion. Al - though Gly12 lo cated next to the -turn re gion is likely to in - crease the back bone flex i bil ity in the -turn re gion, it still re - mains in the well de fined type I -turn mo tif through out the whole 10 ns sim u la tion by form ing two sta ble hy dro gen bonds be tween the am ide and car bonyl groups of res i dues Thr9 and Gly12. It in di cates that interstand hy dro gen bond - ing along with the sta bil ity of the -turn play an im por tant role in keep ing -hair pin in its sta ble na tive-like con for ma - tion. Fig. 7 also shows that some of the side chains are free to fluc tu ate ex cept for Tyr3, Val5, Ile16, and Val18. The hy dro - pho bic in ter ac tions be tween the nonpolar side chains of Tyr3 and Val18, and Val5 and Ile16 re strict the move ment of these side chains, lead ing to con trib ute to the sta bi li za tion of the -hair pin struc ture in pure meth a nol. The pres ent sim u la tion re sult is con sis tent with the pre vi ous NOEs ob ser va tions, in which some res i due side chains lo cated on the same face of the -hair pin were shown to po ten tially sta bi lize the struc ture through the hy dro pho bic in ter ac tion or van der Waals pack - ing. 18 Fig. 5(a) shows that the RMSDs of the back bone C for the -hair pin in pure meth a nol fluc tu ate be tween 0.6 and 1.3 Å dur ing the 10 ns MD sim u la tion. This re sult also strongly in di cates that the struc ture of the -hair pin is very sta ble in the pure meth a nol sys tem. Compared with the sta bi li za tion ef fect in pure meth a - nol as shown in Fig. 7, the -hair pin started to un fold in the 40% (v/v) meth a nol-water sys tem as ob served in Fig. 8. The snap shots at 4 and 6 ns pres ent the two in ter me di ate struc - tures, which show more de vi a tions than the in ter me di ate struc tures sam pled at other time points. Sim i lar re sults can also be seen from Fig. 5(b), in which the RMSD of the back - bone C ver sus MD sim u la tion time was pre sented. The RMSDs var ied be tween 2 and 4 Å dur ing the sim u la tion time course and reached the max i mal value be tween 4 and 6 ns. Pre vi ously, Bonvin et al. 25 con ducted MD sim u la tions of the re duced and ox i dized na tive 19-residue pep tide cor re spond - ing to the first -hair pin of tensamistat and showed that only a lim ited por tion of the ac ces si ble in ter me di ate con for ma tions in the tran si tion state need to be sam pled. In the pres ent study, the -hair pin in the 40% (v/v) meth a nol-water sys tem re veals the pos si ble in ter me di ate con for ma tions in the tran si tion state dur ing the fold ing/un fold ing pro cesses. As can be seen in Fig. 10, the sta ble in ter me di ate con for ma tion sam pled by the -hair pin is the loop-like struc ture main tained through the hy dro pho bic in ter ac tions among the nonpolar side chains of res i dues Val5, Leu7, Ile16, and Val18. The in tro duc tion of wa ter in the 40% (v/v) meth a nol-water sys tem re sults in a strong sol va tion ef fect, lead ing to de struc tion of the am ideamide hy dro gen bonds and con se quently destabilizing the -hair pin struc ture. Mean while, the nonpolar groups of the meth a nol mol e cules in ter act with the nonpolar side chains of res i dues Val5, Leu7, Ile16, and Val18 and sub se quently ori - ent them into the in te rior re gion of these two strands (Fig. 10). The strong hy dro pho bic in ter ac tions among these side chains al low these two -strands to stay close to each other even af ter they have lost most of the interstrand hy dro gen bonds. It has been pre vi ously shown that hy dro pho bic in ter - ac tions among side chains play an im por tant role in sta bi liz - ing a model pep tide con for ma tion in wa ter by ther mo dy - namic and struc tural anal y ses. 51 In the pres ent study, we found that the hy dro pho bic ef fect to sta bi lize the sec ond ary struc ture in so lu tion is more sig nif i cant in the 40% (v/v) meth a nol-water sys tem than in the pure meth a nol sys tem. As can be seen in Fig. 6(b), the to tal num ber of hy dro gen bonds var ied be tween 0 and 5 in the 40% (v/v) meth a nol-water sys - tem dur ing the 10 ns MD sim u la tion. The to tal hy dro gen num ber is usu ally smaller than 3 be tween 4 and 6 ns. The num bers of both interstrand and in side-turn hy dro gen bonds re mained 1 most of the time in the 40% (v/v) meth a nol-water Fig. 8. The snap shots of the -hair pin (grey lines) at (a) 0; (b) 2; (c) 4; (d) 6; (e) 8; (f) 10 ns su per im posed on the crys tal lo - graphic struc ture (blue lines) in the 40% (v/v) meth a nol-water sys tem dur ing the 10 ns MD sim u la tions.

Sta bil ity and Un folding Mech a nism of -Hair pin J. Chin. Chem. Soc., Vol. 50, No. 4, 2003 805 sys tem, which means that about 80 and 50% of the orig i nal hy dro gen bonds have dis ap peared in the strand and turn re - gions, re spec tively. It again in di cates that the -turn re gion is more sta ble than the -strand re gion while -hair pin starts un fold ing in duced by the sol vent with higher po lar ity. Com - paring the pure meth a nol sys tem to the 40% (v/v) meth a nolwater sys tem, we can con clude that both the interstrand and in side-turn hy dro gen bonds play a ma jor role in sta bi liz ing the -hair pin struc ture when it is in its na tive state, whereas hy dro pho bic in ter ac tions among the nonpolar side are re - spon si ble for the in ter me di ate struc ture sta bi li za tion when Fig. 9. The snap shots of the -hair pin (grey lines) at (a) 0; (b) 2; (c) 4; (d) 6; (e) 8; (f) 10 ns su per im - posed on the crys tal lo graphic struc ture (pur ple lines) in the pure wa ter sys tem dur ing the 10 ns MD sim u la tions. -hair pin is in its par tially un folded state. Fig. 9 shows the snap shots of the tar get pep tide in the pure wa ter sys tem dur ing the 10 ns MD sim u la tion. We found that the struc ture dra mat i cally un folded in the so-called zipup man ner (Fig. 1), 25 in which the two -strands opened first, fol lowed by the de struc tion of the -turn re gion. The strong sol va tion free en ergy re quired to re place the am ideamide hy dro gen bonds by the en er get i cally more un fa vor able am ide-water in ter ac tions is com pen sated by the in creased en - tropy dur ing the spon ta ne ous un fold ing pro cess. Fig. 6(c) shows the num bers of hy dro gen bonds ver sus sim u la tion time for -hair pin in the pure wa ter sys tem. The to tal num ber of hy dro gen bonds de creased dra mat i cally. Some re sid ual inter - side chain hy dro gen bonds still can be found dur ing the sim u - la tion, whereas most of the interstrand and in side-turn hy dro - gen bonds dis ap peared. The RMSDs of the back bone C for -hair pin in the pure wa ter sys tem in creased dra mat i cally from 2 Å at the start ing point to about 14 Å at 10 ns (Fig. 5(c)). This again in di cates that the -hair pin un folds rap idly in the pure wa ter sys tem. Com paring Figs. 7-9, we found that the -hair pin is main tained in its na tive state in the pure meth a nol sys tem, whereas it un folds spon ta ne ously fol low ing the zip-up mech a nism 25 (Fig. 1) in the pure wa ter sys tem. It is note wor - thy that the -hair pin par tially un folds and stays in the sta ble in ter me di ate state in which the nonpolar side chains of res i - dues Val5, Leu7, Ile16, and Val18 pro trude into the in te rior of the two -strands and the re sult ing hy dro pho bic in ter ac tions turn the -hair pin into the like-loop struc ture. The pres ent re - sult that meth a nol sta bi lizes the -hair pin in its na tive-state struc ture is con sis tent with the pre vi ous find ings that the na - tive-like -hair pin struc ture can be in duced by re duc ing the sol vent po lar ity by ad di tion of meth a nol or TFE ob served by CD and NMR spec tros copy. 18 The pure meth a nol sys tem is likely to mimic the par tially hy dro pho bic en vi ron ment around the -hair pin pro vided by the sub se quent -he lix in the [2Fe-2S] Ferredoxin I sub unit. It em pha sizes the im por - tance of ad di tional in ter ac tions with other por tions of the polypeptide chain for sta bi liz ing the sec ond ary struc ture and pro mot ing the fold ing of the cor rect ter tiary struc ture of the pro tein. 72-74 Fig. 10. The ste reo view of the loop-like in ter me di ate struc ture of the -hair pin at 6 ns in the 40% (v/v) meth a nol-water sys tem. The hy dro pho - bic in ter ac tions con trib uted by the side chains of Val5, Leu7, Ile16, and Val18 pro trud ing into the in te rior re gion of the two -strands sta bi lize the loop-like in ter me di ate struc ture. EX PER I MEN TAL The ini tial struc ture of the 20-residue pep tide se quence, ASYKVTLKTPDGDNVITVPD (Fig. 3), cor re spond ing to the N-terminal -hair pin from res i due 1 to 20 of the [2Fe-2S] ferredoxin I from the uni cel lu lar blue-green alga A. sa crum

806 J. Chin. Chem. Soc., Vol. 50, No. 4, 2003 Liu and Lin was ob tained from the x-ray crys tal lo graphic struc ture 52 (PDB en try F1XI). There are 8 interstrand hy dro gen bonds in the orig i nal crys tal lo graphic -hair pin struc ture (Ta ble 1). The MD sim u la tions were con ducted in the SGI O200 work - sta tion (Sil i con Graph ics, Inc., Moun tain View, CA, USA)- In sight II (Accelyrs, San Diego, CA, USA) sys tem with the force field Dis cover CVFF (con sis tent va lence force field). 75-77 The ini tial struc ture was first en ergy min i mized by the slow but ro bust steep est de scent method with 5,000 it er a tions to be used as the start ing struc ture for fur ther struc tural com par i - son. Three 35*35*35 Å 3 cu bic lat tices, with the first one con - tain ing 1315 wa ter mol e cules, the sec ond one con tain ing 528 meth a nol mol e cules, and the third one con tain ing 847 wa ter and 211 meth a nol mol e cules, were con structed for the pure wa ter, pure meth a nol, and 40% (v/v) meth a nol-water sys - tems, re spec tively. The start ing struc ture was in jected into the cen ters of these lat tices for 10 ns MD sim u la tions us ing the In sight II pro gram. The time-step used in the MD sim u la - tions was 2 fs. The tem per a tures and pres sure of these sys - tems were main tained con stant by weakly cou pling the tar get pep tide, sol ute, and sol vent, sep a rately, to an ex ter nal tem - per a ture bath at 300 K and to an ex ter nal pres sure bath at 1 atm, re spec tively. 78 All non-bonded in ter ac tions within the 8-Å cut-off ra dius were cal cu lated for each MD time step, while the long-range con tri bu tions, be tween the ra dii of 8 and 14 Å, were up dated ev ery five steps. Po si tion re straints were ap plied on the sol ute heavy at oms dur ing the equi lib rium stage of the sim u la tion to al low the sol vent (e.g., wa ter and meth a nol) to equilibrate with out dis turb ing the tar get pep tide struc ture. The tra jec to ries and co or di nates of the pep tide mol - e cule were saved ev ery 100 ps for fur ther anal y sis. All sim u - la tions were per formed on an SGI O200 sys tem with 64-bit HIPS RISC R12000 270 MHz CPU and a PMC-Sierra RM7000A 350 MHz pro ces sor. About 1s of CPU time was re quired for one 2 fs sim u la tion, lead ing to a to tal run ning time of 120 to 130 CPU days for each of the three sys tems. Al though some com pli cated al go rithms have been pro - posed to mea sure the structural similarity be tween pro teins, 79,80 the root-mean-square de vi a tion (RMSD) re mains the most com monly used. For each MD sim u la tion, the RMSDs of the tra jec to ries re corded ev ery 100 ps in ter val were cal cu lated for the back bone C atom of the -hair pin dur ing the course of 10 ns MD sim u la tions at 300 K with ref er ence to the start - ing struc ture, which has been pre vi ously en ergy min i mized. 81 As a re sult, RMSD is a use ful mea sure of struc tural sim i lar ity for closely re lated pro teins 82 or for the struc tural change dur - ing pro tein fold ing/un fold ing pro cesses. The RMSDs were cal cu lated af ter op ti mal su per im po si tion of the co or di nates to re move translational and ro ta tional mo tion. 83 The start ing struc ture of the MD sim u la tions, which was gen er ated from about 10 ps equil i bra tion, gen er ally had a very sim i lar fold - ing struc ture to that of the ini tial crys tal lo graphic struc ture with RMSD 0.2 Å (data not shown). The hy dro gen bond ing was pre dicted based on the Kabsch-Sander al go rithm, 84 in which pat tern rec og ni tion of hy dro gen bonds was cor re lated to the geo met ri cal fea tures. The de fault hy dro gen bond ing en ergy cri te rion of -0.5 kcal/mol was used. AC KNOWL EDGE MENTS The au thors thank the Na tional Sci ence Coun cil of Tai - wan for fi nan cial sup port (pro ject num ber NSC-89-2214-E- 027-006). We also thank Kauo-Cheng Liu and Nan-Chen Ten of NTUT for data anal y sis. Ab bre vi a tions H-bond, hy dro gen bond; MD, mo lec u lar dy nam ics; RMSD, root-mean-square de vi a tion. 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