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1 Title Studies on the Refolding Proess of Dissertation_ 全文 ) Author(s) Onda, Maki Citation Kyoto University ( 京都大学 ) Issue Date URL Right Type Thesis or Dissertation Textversion author Kyoto University

2 STUDIES ON THE REFOLDING PROCESS OF OVALBUMIN MAKI ONDA 1999

3 CONTENTS ~ABBREVIATIONS INTRODUCTION 1-2 ~CHAPTER I Refolding Proess of Disulfide-bonded 3-25 and redued Ovalbumin from Urea-denatured States ~CHAPTER II Refolding of Urea-denatured Ovalbumin Comprises Non-Native Disulfide Isomers ~CHAPTER III Charaterization of Refolding Intermediate 45-7 of Ovalbumin Using Non-Native Disulfide Isomers SUMMARY i\cknowledgement 73 JREFERENCES ]PUBLICATIONS 79

4 ABBREVIATIONS INTRODUCTION CD Tris EDTA DTI IAEDANS HPLC SH ondition SS ondition IAA ANS irular dihloism tris(hysroxymethyl)-aminomethane ethylenediamine tetraaetate dithiothreitol N-iodoaetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine high performane liquid hromqtography disulfide-redued ondition disulfide-bonded ondition iodoaeti aid anilino-1-naphthalene-8-sulfonate Protein struture is onstruted to fold a polypeptide han spontaneously. Folding does not, however, our by sampling all possible onformations randomly until the one with the lowest free energy is enountered (1). There are pathways during protein folding. To analyze the mehanism and pathway of folding is very important to understand the priniple of protein struture onstrution. A disulfide protein is a useful model for the investigation of protein folding mehanisms (2). Disulfide proteins that assume fully unfolded (3-12) or partially unfolded onformation (13-15) in their disulfideredued states an be oxidatively refolded with the aid of a hemial oxidant. The kineti folding pathways of some small single-domain proteins, suh as bovine panreati trypsin inhibitor (3-7), ribonulease A (8), ribonulease T1 (9), a-latalbumin (1), and hirudin (11, 12) have been eluidated by identifying the disulfide strutures in partially disulfidebonded intermediates. A seond lass of disulfide proteins that assume a native-like onformation in the disulfide-redued state has also been the target of protein folding studies. The influene of protein onformation on disulfide bond formation in the onstant fragment of immunoglobulin light hain (16) and onversely, the influene of disulfide bonds in the folding kinetis of growth hormone (17), have been demonstrated. Ovalbumin has unique strutural harateristis as an alternative model for the seond lass of disulfide proteins. This egg white protein ontains four ysteine sulfhydryls along with an intrahain disulfide in a single 1

5 polypeptide hain (18, 19). In urea-denatured state near neutral ph, intrahain sulfuydryl/disulfide exhanges our and produe fifteen disulfide isomers theoretially (2). Furthermore, the egg white protein an refold to native state without reover of the native disulfide under the disulfide-redued ondition (21). These harateristis enable to analyze the refolding pathways and intermediates with traing disulfide isomers without using oxidizing agents. A major problem enountered in the kineti analysis of an oxidative refolding pathway is related to protein sulfuydryl aessibility to an oxidizing agent in the first step. In this study, the author attempted to eluidate the refolding proess of ovalbumin with analysis for intrahain disulfide rearrangements and disulfide isomers. In hapter I, two types of intermediates are deteted during refolding proess, and the author demonstrates the refolding pathway by means of kineti analysis. In hapter II, it is delared that urea-denatured ovalbumin omprising non-native disulfide isomers orretly refold to native protein via sulfuydryl/disulfide exhange reations. In hapter III, refolding intermediate is haraterized by use of hemial modified ovalbumin with a non-native disulfide Cys 367- Cys 382, furthermore, the author disusses about refolding proess and intramoleular interation govern the protein struture onstrution. CHAPTER I Refolding Proess of Disulfide-bonded and redued Ovalbumin from Urea-denatured States Ovalbumin ontains four ysteine sulfuydryls (Cys 11, Cys 3, Cys 367 and Cys 382 ) along with an intrahain disulfide (Cys 73-Cys 12) in a single polypeptide hain of 385 amino aid residues (18, 19). It has been shown that the onformational state of the disulfide-redued ovalbumin is almost indistinguishable from that of the disulfide-bonded form (22). Furthermore, the egg white protein an refold from urea-denatured state in the SH ondition, indiating spontaneous protein folding without the native disulfide bond (21 ). When disulfide-bonded ovalbumin denatured in a high onentration of urea is plaed in non-denaturing onditions, the protein refolds muh less effiiently than the disulfide-redued form does (21 ). This may be related to the observation that many non-native disulfide isomers that have one disulfide and four sulfuydryls in a moleule are produed in a high onentration of urea (2). The detailed refolding mehanism, however, remains to be investigated for both disulfide-bonded and disulfide-redued ovalbumin. In the this hapter, the author investigated the refolding mehanism of ovalbumin in SS and SH onditions using the denatured protein state, D A, as the starting protein sample. D A was produed by inubation of the disulfide-bonded and disulfide-redued ovalbumin in 9 M urea at ph 2.2, where possible sulfuydryl/disulfide exhange reations in the disulfidebonded protein are almost ompletely bloked ( 4). Refolding was 2 3

6 initiated by a ph jump proedure in whih the denatured protein D A is plaed in a refolding buffer with near neutral ph. The author reports here that a partially folded intermediate state, IN, is formed very rapidly in either the SS or SH ondition as deteted by far UV CD and intrinsi tryptophan fluoresene spetra. After the initial burst phase, the time ourse for the onformational regain followed biphasi kinetis in the SS ondition, but it followed monophasi kinetis in the SH ondition. Aording to the data from a peptide mapping analysis, the native disulfide in D A undergoes nonspeifi disulfide rearrangements at an early refolding stage in the SS ondition and then is reovered during the subsequent slow refolding. These data along with other findings are onsistent with a refolding mehanism for ovalbumin whih inludes nonprodutive side hain-side hain interations in the early intermediate IN, whih requires strutural reorganization for subsequent orret refolding. Denaturation and Refolding of Ovalbumin For the refolding in the SS ondition, denatured ovalbumin D A was prepared by inubating the native, disulfide-intat protein at 1. mg/ml, at 37 C for 3 min in.25 M HCl ontaining 1. mm Na-EDTA and 9 M urea, ph 2.2. Refolding was initiated at 25 o by 2-fold dilution of D A with buffer A (5 mm Tris-HCl buffer, ph 8.6, 1. mm Na-EDTA) giving a final ph value of 8.2. The proteins were allowed to refold at 25 o and were then analyzed by intrinsi tryptophan fluoresene and CD spetrum. The buffers were degassed at redued pressure and equilibrated under N 2 atmosphere prior to the refolding. An equilibrium intermediate la was produed by 2-fold dilution of D A with 5 mm potassium phosphate buffer, ph 2.2, ontaining 1. mm Na-EDTA. The equilibrium intermediate was plaed for refolding in a near neutral ph ondition, ph 8.2, by being mixed with.2 volume of 3. M Trizma (Tris) base. For the experiments in the SH ondition, the native disulfide In EXPERIMENTAL PROCEDURES ovalbumin was fully redued at 1 mg/ml by inubation with 15 mm Materials Ovalbumin (A 1 -ovalbumin, diphosphorylated form) was purified from fresh egg white by rystallization in an ammonium sulfate solution and subsequent ion exhange olumn hromatography as desribed (23, 24). Diphenylarbamyl hloride-treated trypsin (type XI) and hymotrypsin (type II) were purhased from Sigma. Ahromobater proteinase I (EC ) was obtained from Wako Pure Chemial Industries. dithiothreitol at 37 o for 2 h in 5 mm Tris-HCl buffer, ph 8.2, ontaining 1. mm Na-EDTA (22). Disulfide-redued DA was produed by 1-fold dilution of the native, disulfide-redued protein with.25 M HCl ontaining 1. mm Na-EDTA and 9 M urea, ph 2.2. The refolding from D A, the preparation of la, and the refolding from la in the SH ondition were arried out in the same ways as in the SS ondition, exept that all of the diluents ontained.25 mm dithiothreitol. 4 5

7 Measurement of Intrinsi Tryptophan Fluoresene dihlorophenolindophenol and L-asorbate (25) was 2 ms. The fluoresene spetrum of ovalbumin was measured with a fluoresene spetrophotometer (Hitahi, model F-3). The intrinsi tryptophan residues in ovalbumin were exited at 295 nm, and emission spetrum was reorded at a wavelength range from 3 to 42 nm. All measurements were arried out at a onstant temperature of 25 C. The time ourse of fluoresene intensity hange was monitored at 338 nm emission. For the spetrum measurements at an early refolding time, the time ourse of fluoresene intensity hanges was monitored at various emission wavelengths with exitation at 295 nm, and data at a refolding time of 1 s were plotted. Differential Sanning Calorimetry D A was refolded for 2 h under the SS and SH onditions, onentrated about 15-fold using a onentrator (Amion, Centriprep-1), and passed through a Sephadex olumn (NAP-1, Pharmaia Bioteh In.) equilibrated with 1 mm sodium phosphate buffer, ph 6.. Overall reoveries from the original D A forms were about 7%. The refolded proteins and orresponding native protein ontrols were analyzed with a differential sanning alorimeter (Miro Cal, MCS-DSC). The protein onentration was.4 mg/ml in 1 mm sodium phosphate buffer, ph 6.. The CD Spetrum Measurement The far UV CD spetrum was reorded at 25 o with a spetropolarimeter (JASCO, J-72). The CD data were expressed as mean residue elliptiity (degrees m 2 /dmol) by using 111 a s the mean residue weight of ovalbumin. CD spetra at a short refolding time were determined by measuring the time-dependent inrease in CD elliptiities at various wavelengths, and the values at the refolding time 1 s were plotted as a funtion of wavelength. For rapid mixing experiments, D A was applied to a stopped-flow rapid kinetis aessory (Applied Photophysis, RX.1) attahed to the same spetropolarimeter, and hanges in CD elliptiity at 23 nm were reorded at 25 o after an 11-fold dilution with buffer A. The mixing dead time that was determined by using 2,6- temperature was sanned at 1 K min- 1 Analyses for Disulfide-involved Half-ystines At various refolding times in the SS ondition, sulfhydryl/disulfide exhanges were quenhed by mixing the protein samples with.24 volume of 2 M HCI. Disulfide-involved half-ystines were determined by alkylation with a fluoresent reagent N-iodoaetyl-N9-(5-sulfo-1- naphthyl)ethylenediamine ; IAEDANS, and a subsequent peptide mapping proedure as desribed (2). Briefly, the aid-quenhed protein was neutralized and alkylated with.1 M iodoaetamide in 9 M urea solution. The alkyl a ted protein was preipitated in a old aetone-hcl solution, dissolved in a urea solution, redued with dithiothreitol, and then modified with IAEDANS. The modified protein was extensively proteolyzed with 6 7

8 the ombination of trypsin, hymotrypsin, and Ahromobater protease I. The resultant peptides were analyzed by reversed phase HPLC with fluoresene monitoring (exitation, 34 nm; emission, 52 nm). For the standard experiment, the intat ovalbumin was fully redued with dithiothreitol, all ysteine residues were labeled with IAEDANS, and the moleule was proteolyzed in the same way. Q.) {.) 1 8 ~ 6 en Q.) '- :::J 4 Ll.. N (1) (.) (1) (.) C/) (1) '- ::J u_ Wavelength(nm) RESULTS 2 Folding Intermediate Deteted by Optial Methods Ovalbumin ontains three tryptophan residues, Trp 148 in helix F, Trp 184 as the nearest neighbor residue of the COOH terminus of strand 3A, and Trp 267 in helix H (26). The onformational states of different forms of ovalbumin were analyzed by the intrinsi tryptophan fluoresene spetrum. As shown in Fig. 1, the fluoresene emission spetrum of native ovalbumin was indistinguishable for the disulfide-redued and disulfide-bonded forms; the native proteins showed an emission maximum at 338 nm. D A showed a typial red shift spetrum of an unfolded protein; the emission maximum was shifted to a longer wavelength of 352 nm, and the fluoresene intensity was dereased to 32% of the native form. When D A was refolded at a near neutral ph, the protein showed at the early refolding time of 1 s a fluoresene spetrum that had a peak at the same wavelength but with muh dereased intensity (57%) ompared with the spetrum of native ovalbumin Wavelength(nm) FIG.l. Fluoresene emission spetra of various states of ovalbumin. In the SS (solid lines) and SH (dotted lines) onditions, the tryptophan residues in various states of ovalbumin were exited at 295 nm, and the emission spetra were reorded at 25 o as desribed under "Experimental Proedures." N (thik lines) represents native ovalbumin. D A (thin lines) and DN (thik lines) are the proteins denatured by protein inubation in 9 M urea at 37 o for 3 min at ph 2.2 and at ph 8.2, respetively. The equilibrium intermediate state la (thin lines) was produed by 2-fold dilution of D A with a non-denaturing buffer, ph 2.2. The intrinsi tryptophan fluoresene of the proteins refolded from D A for 5 min (5 M, thin lines) and for 2 h (2H, thin lines) was reorded in the same way. For the fluoresene spetra at an early refolding time (IN ), the time ourse of fluoresene hanges was reorded during refolding in the SS (open irles with thin solid line) and SH (losed irles with thin dotted line) onditions at different emission wavelengths, and the values at the refolding time of 1 s were plotted as a funtion of emission wavelength. In the inset the fluoresene spetra at ph 8.2 of the native protein (thik solid line) and the protein denatured in 9 M urea for 1 s (open triangles with a thin line) and for 5 and 3 min (thin lines from top to bottom) were reorded in the same way. The fluoresene intensity is shown by an arbitrary unit. 8 9

9 The fluoresene intensity was then inreased at 5 min of the refolding and reahed more than 9% of the value for the native protein at a prolonged refolding time of 2 h. The blue shifted spetrum observed at the early refolding time ould be aounted for not by a solvent effet of dereased urea onentration, but rather by a hange in protein onformation, sine the maximum wavelength of fluoresene was the same 338 nm at the early stage (1 s) of the urea-indued denaturation of ovalbumin (Fig. 1, inset). The same approah was arried out using far UV CD spetrum as a onformational probe. As shown in Fig. 2, native ovalbumin again showed almost the same spetrum in the SS and SH onditions. At the early stage (1 s) of the refolding in the SS and SH onditions, ovalbumin showed a CD spetrum that had 6% absolute elliptiity at 222 nm of the native form. The absolute elliptiity inreased further with time of refolding, and the CD spetra of the proteins refolded for a prolonged time of 2 h were almost exatly the same as those of the native proteins. When the time ourse of CD elliptiity hanges in the SS ondition was monitored using a rapid mixing tool, the obtained data, while noisy, were onsistent with that IN formation from D A is finished within the mixing dead time of 2 ms (Fig. 2, inset). The same was also true for the IN formation in the SH ondition (data not shown). These data indiated that the partly folded intermediate IN is formed very rapidly as an initial burst during the refolding in the SS and SH onditions at the near neutral ph; the intermediate had strutural harateristis of 6% absolute CD elliptiity at 222 nm and 57% fluoresene intensity at 338 nm of the native form. Figs. 1 and 2 also demonstrate that D A was transformed by 2-fold dilution with potassium phosphate buffer, ph 2.2, into a partially folded form la. T"" I -2 E -4 "' C\1 E -6 () > (]) -8 "'..._... M -1 >< 'or-,..., ~ N.5 Time(s) Wavelength(nm) FIG.2. Far UV CD spetra. In the SS (solid lines) and SH (dotted lines) onditions, the far UV CD spetra in various states of ovalbumin were reorded at 25 o as desribed under "Experimental Proedures." The designations of N, D M IN, IA, 5M, and 2H for the different ovalbumin states are the same as in Fig. 1. For the CD spetra for early refolding intermediates, the time ourse of hanges in CD elliptiities at different wavelengths were reorded during refolding in the SS (open irles with thin solid line) and SH ondition (losed irles with thin dotted line), and the values at the refolding time of 1 s were plotted as a funtion of wavelength. In the inset, the urea-denatured disulfidebonded D A was refolded at ph 8.2, and the hanges in CD elliptiities at 23 nm were reorded for 1 s using a stopped-flow tool as desribed under "Experimental Proedures"; 23 nm was employed beause of less noisy CD elliptiities at this wavelength than at a peak wavelength of 222 nm. D and N shown by arrows represent the CD elliptiities at 23 nm of D A and of the native protein, respetively

10 The far UV CD spetrum of la was almost indistinguishable from that of the early folding intermediate IN. The fluoresene spetrum of la showed the maximum at the same wavelength (338 nm) as that of IN. The dereased fluoresene intensity for la an be aounted for by solvent effets by aid, sine D A shows dereased fluoresene intensity ompared with the urea-denatured ovalbumin at ph 8.2 (Fig. 1 ). In addition, a previous report has shown that native ovalbumin represents signifiantly dereased fluoresene (about 55%) at ph 2.2 from that at ph 8.2 but almost indistinguishable far UV CD spetra at the two different ph values (27). The disulfide rearrangements should be almost ompletely bloked at ph 2.2 ( 4). Furthermore, the onformational state was onstant during 2-h inubation at ph 2.2, as evaluated by the far UV CD analysis (data not shown). The author therefore onluded that the aid-quenhed la is an denaturation temperatures were 77.7 o for the native protein and 77.6 o for the refolded protein. Likewise, the melting temperature for the protein refolded in the SH ondition (7.8 C) was almost the same as that for native, disulfide-redued ovalbumin (7.7 C). The minor peak observed for the refolded protein in the SH ondition may be aounted for by reoxidation of the native disulfide during the refolding time of 2 h and/or during the protein onentration proedure prior to the alorimetry analyses, sine the peak temperature was about 77 C, whih is essentially the same value as the melting temperature for the native, disulfide-bonded protein. The peak area was, however, less than 8% of the major peak. The author therefore onluded that most of the ovalbumin moleules an refold orretly in the SS and SH onditions into the same protein energy states as the native proteins. equilibrium intermediate state that possesses strutural harateristis equivalent to IN. Differential Sanning Calorimetry Analysis of Refolded Ovalbumin The data from the intrinsi fluoresene and far UV CD analyses showed that most if not all ovalbumin moleules refold into the native state at 2 h inubation (Figs. 1 and 2). The integrity of native refolding was investigated more rigorously by an alternative method of differential sanning alorimetry. Fig. 3 demonstrates that the protein refolded for 2 h in the SS ondition underwent thermal transition with almost the same melting temperature as did the native protein ounterpart; the thermal FIG.3. Differential sanning alorimetry analysis of refolded ovalbumin. Ovalbumin refolded from D A for 2 h and native protein ontrols were analyzed by differential sanning alorimetry in 1 mm sodium phosphate buffer, ph 6., with a temperature sanning rate of 1 K min- 1 Endothermi transition profiles for the native, disulfide-bonded protein (NSS), for the native, disulfide-redued protein (NSH), for the protein refolded in the SS ondition (RSS ), and for the protein refolded in the SH ondition (RSH) are arbitrarily shifted on the ordinate sale for larity E C'd () ~ -a. 5 () Temperature CC)

11 ~ Refolding Kinetis after the Initial Burst Phase The data in Figs. 1 and 2 also show that slow refolding proeeds after rapid formation of the intermediate state. The time ourses of the refolding from D A after the initial burst were examined by intrinsi fluoresene and CD analyses. Fig. 4 (panels A and B) shows the timedependent inreases in the fration of the re-folded protein after 6 s of the refolding from D A The refolding in the SS ondition apparently followed biphasi kinetis (for the quantitative alulation, see "Disussion"). In ontrast, ovalbumin refolded with simple monophasi first-order kinetis in the SH ondition. No signifiant differene was observed in either the SS or SH ondition for the kineti profiles obtained by the fluoresene and CD analyses. The data in Figs. 1 and 2 demonstrate that D A is transformed by dilution with potassium phosphate buffer, ph 2.2, into a partially folded form la, whih has strutural harateristis equivalent to those of IN. To examine whether or not la an refold in a manner similar to the refolding from D A, the aid-quenhed equilibrium intermediate was plaed in a near neutral ondition, and the time ourse for the refolding was monitored by intrinsi fluoresene and CD analyses. As shown in the panels C and D of Fig. 4, la refolded with essentially the same kinetis as D A; the refolding followed biphasi kinetis in the SS ondition but monophasi kinetis in the SH ondition _ z LL E ' A.2 LL 8...> o +'"" z ' '"".6 (.) o.4 '-.4 LL Refolding Time (min) FIG.4. Time ourse for the refolding after the initial burst phase. D A and IA were prepared as desribed under "Experimental Proedures". The time ourse for the refolding at ph 8.2, 25 o from D A (panels A and B) and from IA (panels C and D) in the SS (open irles) and SH (losed irles) onditions were monitored by the intrinsi tryptophan fluoresene at 338 nm (panels A and C) and by CD elliptiity at 222 nm (panels B and D) as desribed under "Experimental Proedures". The ordinate shown by FN (t) represents the fration of the native form at the refolding time t, whih was alulated by the equation FN (t) = (X - X, )/(X - XN ), where X and X, are the values at the refolding times of 6 s and t, respetively. XN is the final value of the refolding; the fluoresene intensity and CD elliptiity at the refolding time of 2 h were employed as XN. As ontrol experiments, disulfide-bonded and disulfide-redued ovalbumin preinubated at ph 2.2 in the absene of urea were diluted 2-fold with buffer A giving the same final ph value of 8.2; the CD and fluoresene intensities were onfirmed to show invariably the native values during the inubation time of 12 min. The solid urves represent nonlinear least squares fits of the dupliate experimental data to theoretial rate equations (Equations 1 and 2, desribed under "Disussion"). For the nonlinear least squares fits, a program utilizing a Levenberg-Marquardt algorithm was employed

12 Sulfhydryl/Disulfide Exhanges during Refolding The data in Fig. 4 indiate that the biphasi kinetis are lose! y related to the presene of the intrahain disulfide bond. The denatured state D A should ontain the native disulfide Cys 73-Cys 12 and four ysteine residues Cys 11, Cys 3, Cys 367 and Cys 382 sine intrahain sulfhydryl/disulfide exhange reations possible for fully denatured ovalbumin (2) should be almost omplete! y quenhed at the employed ondition of ph 2.2 ( 4). The question, however, arose as to whether the involvement of disulfide rearrangement reations during the refolding at ph 8.2 results in the more omplex kinetis in the SS ondition than in the SH ondition. To examine the possibility of disulfide rearrangements during the refolding, the author determined the disulfide-involved halfystines by the peptide mapping proedure at various refolding times. As shown in Fig. SA, only Cys 73 and Cys 12 were deteted as disulfideinvolved ysteines at the refolding time from D A. The disulfideinvolved Cys 73 and Cys 12, however, dereased signifiantly at an early refolding time of 1. min; onomitantly at this stage, all of the other four ysteines, Cys 11, Cys 3, Cys 367 and Cys 382, were deteted as the disulfide-involved ysteines. The amounts as disulfide-involved ysteines were the minima (about 6%) for Cys 73 and Cys 12 but the maxima (about 2%) for the other ysteines at 1S min of the refolding. The disulfide-involved Cys 73 and Cys 12, however, both inreased gradually after 1S min, and the amounts were about 9% at 2 h refolding. The next question was related to the refolding stage that involves the nonprodutive disulfide rearrangements. In the refolding system from D A' ovalbumin denatured under aid-urea onditions was plaed in near neutral buffer onditions to be allowed to refold. The original D A, therefore, may first be transiently transformed by ph jump to the highly denatured state at ph 8.2 and then to the early intermediate state IN by onformational onversion. Suh a highly denatured state should undergo extensive disulfide rearrangements at near neutral ph (2). However, the data in Figs. 1, 2 and SA showing that the rapid formation of IN preedes the disulfide rearrangement appeared to support the rearrangement reations in IN rather than in the transient denatured state. To onfirm this, the author analyzed disulfide-involved ysteines during the refolding from IA, whih has the native disulfide and equivalent strutural harateristis to IN, sine the involvement of ovalbumin in the transient denatured state an be skipped in this refolding system. The data in Fig. SB demonstrate that the disulfide-involved Cys 73 and Cys 12 dereased at an early refolding time in almost the same manner as in the diret refolding from D A. The disulfide-involved Cys 73 and Cys 12 showed the minima at 1S min and S min, respetively, and then both inreased gradually; at 2 h of the refolding, the amounts again reahed values of about 9%. Almost the same time ourses as in the diret refolding from D A were also observed for the other four ysteine residues; the disulfide-involved Cys 11, Cys 3, Cys 367 and Cys 382 inreased tentatively at an early refolding time and then dereased with the time of the refolding

13 '(i)... (/) >- ' > > I ' \;:::: ::::J (/)... :;::; C'O a: 1. A LOEJ.6 LOEJ s.8 s.8 z.4 z.4 UJ.6 UJ.6 UJ UJ.4 Ll Ll Time (min) Time (min) Refolding Time (min) FIG.S. Disulfide rearrangements during the refolding. In panel A, D A prepared as desribed under "Experimental Proedures" was refolded at ph 8.2, 25 C. In panel B,IA that had been prepared by 2-fold dilution of D A with potassium phosphate buffer, ph 2.2, was refolded at 25 o after being mixed with a.2 volume of 3 M Tris base, giving a final ph value of 8.2. At various refolding times in the SS ondition, the disulfide-involved ysteines for Cys 11 (open squares), Cys 3 (losed squares), Cys 73 (losed irles), Cys 12 (open irles), Cys 367 (losed triangles), and Cys 382 (open triangles) were determined using a peptide mapping analysis (2). The data are the averages for dupliate determinations. The insets present the theoretial time ourses for FN-ss (t) whih is defined as the fration of the ovalbumin speies with the native disulfide Cys 73-Cys 12; FN-ss (t) was alulated using Equation 3 and the refolding rate onstants in Table 1 desribed under "Disussion." The solid and dotted urves were obtained by alulation using the rate onstants from the fluoresene and CD analyses, respetively. The theoretial urves for the refolding from DA (panel A) show the minimum values at 9. and 1.5 min for the fluoresene and CD data, respetively. For the refolding from la (panel B), FN-ss (t) was the minimum at 1.3 min for the fluoresene data and at 9.2 min for the CD data. DISCUSSION The data in the present study demonstrate that most, if not all, of the ovalbumin moleules in D A refold orretly in either the SS or SH ondition into the native form, N, as evaluated by the intrinsi fluoresene and far UV CD spetra (Figs. 1 and 2). Furthermore, the results from the differential sanning alorimetry analysis provide evidene for the equivalent onformational stability of the refolded protein to the native protein ounterpart (Fig.3). To my knowledge, this is the first demonstration that the integrity of native refolding was onfirmed by differential sanning alorimetry for suh a large protein as ovalbumin. An intermediate state IN is produed from D A at an early stage (within 2 ms) of the refolding at ph 8.2 (Figs. 1 and 2) in either the SS or SH ondition. An equivalent equilibrium intermediate IA is formed when D A is diluted with a non-urea aidi buffer (Figs. 1 and 2). As unexpeted findings, the native disulfide Cys 73-Cys 12 undergoes nonprodutive disulfide rearrangements due to sulfhydryl/disulfide exhanges with the other four ysteines during the refoldings from D A and IA in the SS ondition (Fig. 5). These data are onsistent with the following Sheme for the refolding proess of urea-denatured ovalbumin, (ph8.2) (Sheme 1) (ph2.2)

14 where IN[ non-n] represents the mispaired-disulfide intermediate that is formed during the refolding in the SS ondition. IN[3-4] is the ondition and to Equation 2 in the SH ondition. The first-order rate onstants obtained by the data fitting analysis are summarized in Table 1. intermediate with the native disulfide Cys 73-Cys 12 (SS ondition) or without any intrahain disulfide (SH ondition) and assumed to be the ompetent one for subsequent folding into N[3-4] with a first-order rate onstant, kf. Through the disulfide rearrangements, IN[non-N] undergoes reversible interonversion with IN[3-4] ; k+ 1 and k_ 1 are the first-order rate onstants for the onversions from IN[non-N] to IN[3-4] and from IN[3-4] to IN[non-N], respetively. The small arrows with thin solid lines represent the initial burst for the onformational transition and/or ph jump steps. Sheme 1 is onsistent with the kineti data of the refoldings after the initial burst phase (Fig. 4). FN (t), whih is defined as the fration of N[3-4] at a refolding time t, an be expressed in the SS ondition as follows. TABLE 1 Rate onstants impliated for the ovalbumin refolding The first-order rate onstants for Sheme 1 were obtained by fitting the refolding data in Fig. 4 to Equation 1 in the SS ondition and to Equation 2 in the SH ondition. Refolding Conditions Conformational from probea DA ss FL la ss FL DA SH FL la SH FL DA ss CD la ss CD DA SH CD la SH CD Rate onstants (min' 1 ) k. 1 k_ 1 kr a FL and CD represent the fluoresene and CD spetrum analysis, respetively (Eq. 1) where r 1 and r 2 are related to the rate onstants as r 1 = -a +J a 2 - ~, r 2 = -a -J a 2 - ~, 2a = k+ 1 + k_ 1 + kf, and ~ = k+ 1 kf. In the SH ondition, no disulfide rearrangements are involved (k+ 1 = k_ 1 = ). Equation 1 an therefore be redued into a single exponential first-order rate equation. (Eq. 2) As displayed by the solid lines in Fig. 4, the data for the refolding from either D A or IA an be fitted quite adequately to Equation 1 in the SS The three first-order rate onstants eah show, in either the SS or SH ondition, almost equivalent values for the two refolding systems (from D A and la) and for the two onformational probes, reinforing the present refolding mehanism of Sheme 1. The obtained rate onstants appear also reasonable for the eluidation of the time ourse for the disulfide rearrangements in Fig. 5. Aording to Sheme 1, FN-ss(t), whih is defined as the ovalbumin fration with the native disulfide at refolding time t, should orrespond to the sum of the frations of N and IN. FN-ss (t) an therefore be expressed as follows

15 FN-ss(t) = 1- exp(r 2 t) (Eq. 3) The fration of ovalbumin speies with the native disulfide an therefore be alulated as a funtion of t using the rate onstants in Table 1 as displayed in the insets of Fig. 5. The value for the disulfide-involved Cys 73 and Cys 12, whih also inludes their partiipation in mispaired disulfides with the other ysteines, should not be exatly the same as, but should be losely related to, the amount of the protein speies with the native disulfide Cys 73-Cys 12. Fig. 5 represents that the theoretial urve is ompatible with the experimental data at the qualitative level. The values for FN-ss (t) derease rapidly at early refolding times showing the minima at 9-1 min and then gradually inreasing with the time of the refolding. Suh theoretial profiles were very similar to the experimental time ourses for the disulfide-involved Cys 73 and Cys 12, whih show the minima at 5-15 min. The results from the data-fitting analyses provide important information about the refolding mehanism of ovalbumin. First, the refolding of ovalbumin follows biphasi kinetis in the SS ondition (Fig. 4). As a general mehanism for biphasi refolding kinetis, the involvement of the parallel pathway that is related to is-trans isomerization of proline residues has been demonstrated (28). Ovalbumin indeed ontains 14 proline residues. Sheme 1 is, however, onsistent with the biphasi kinetis in the SS ondition without inluding parallel pathways. A deelerated folding mehanism in disulfide-bonded ribonulease T1 whih IS aounted for by dereased proline isomerization rate (29) is also unlike! y in the refolding of ovalbumin, sine the folding rate onstant kf is almost idential in the SS and SH onditions (Table 1 ). It is, therefore, very like I y for the refolding kinetis of ovalbumin that the effets of the proline isomerization problem, if any, work in an undifferentiated manner in the SS and SH onditions. Seond, aording to the author's previous simulation analysis for the disulfide rearrangements in ovalbumin, the rate onstant k for a sulfhydryl/disulfide exhange in 9 M urea at ph 8.2 is related to the equation k = 82 ne - 2, where ne is the "effetive number" of amino aids separating a disulfide and a relevant sulfhydryl (2). For the initial disulfide rearrangements by the nuleophilli attaks of the four ysteines on the native disulfide Cys 73-Cys 12, the rate onstants should be in the order Cys 3 > Cys 11 > Cys 367 > Cys 382 ; the absolute rates for the produtions should be quite variable from the most rapid reation (attak by Cys 3 forming Cys 3-Cys 73 and Cys 3-Cys 12 ) of.44 min 21 to the slowest reation (attak by Cys 382 forming Cys 73-Cys 382 and Cys 12-Cys 382 ) of.12 min- 1 As shown in Table 1, the rate onstant for the formation of mispaired disulfides in IN (k_ 1 ) is approximately half of that for the most rapid reation in the highly denatured state. Although the value for k_ 1 is an average value for many rearrangement reations, the data in Fig. 5 represent almost equivalent disulfide partiipation for Cys 11, Cys 3 and Cys 382, exept for a slightly lower value for Cys 367. These data may reflet some ompat but highly flexible onformational state for IN in whih the effetive onentrations of

16 the four ysteines sulfhydry Is relative to the native disulfide are all signifiantly high. The present refolding mehanism of ovalbumin has impliations for the urrent view of the nature of protein folding intermediates. The equilibrium molten globule state has been observed with several globular proteins as a partially denatured form; the state has been laimed to be a general produtive intermediate in protein folding (3-32). There are, however, examples in whih protein folding proeeds via two-state behavior without an apparent folding intermediate (33, 34). Theoretial approahes for protein folding have suggested that folding intermediates aumulate beause of kineti traps aused by partial misfolding (35, 36). Indeed, experiments with ytohrome represent that the refolding proeeds via an essentially two-state behavior without any apparent intermediate and that a trapped intermediate under three-state onditions results from misfolding of the polypeptide hain in the initial ollapse step (37). In the ovalbumin refolding, IN is formed as an initial burst, and the subsequent regain of the native onformation proeeds slowly (Figs. 1, 2 and 4). The observations that both the native disulfide speies D A and la undergo non-native disulfide rearrangements in an early stage of the refolding (Fig. 5) learly demonstrate that nonspeifi side hain-side hain interations are involved in an early intermediate of ovalbumin refolding. The early intermediate IN is therefore very likely to be a moleular ollapse The data in Fig. 5 represent that the protein speies with a mispaired disulfide an refold orretly into the native disulfide form via disulfide rearrangements. A sulfhydryl/disulfide protein that ontains, in its natural or engineered form, both ystine and ysteine residues in the native moleule as in ovalbumin may generally work as a useful model for the investigation of a protein folding mehanism. The folding mehanisms of disulfide proteins have been investigated using mostly oxidative refolding systems in whih the disulfide-redued, denatured state is transformed into the native, disulfide-bonded form with the help of an oxidizing agent. A protein disulfide formation, however, inludes multiple hemial steps: the first step is the intermoleular attak of a protein sulfhydryl on an oxidizing disulfide agent generating a protein mixed disulfide, and the seond step is the intramoleular attak of a seond protein sulfhydry 1 on the mixed disulfide (2). Intrahain sulfhydryl/disulfide exhanges also our during the refolding of most of the small single-domain proteins (3-12). A major problem enountered in the kineti analysis for an oxidative refolding pathway is related to the protein sulfhydryl aessibility in the first step to an oxidizing agent, as demonstrated for bovine panreati trypsin inhibitor (17). In the refolding proess of the sulfhydryl/disulfide protein, however, only intrahain sulfhydryl/disulfide exhange reations should be inluded. The sulfhydryl aessibility problem in the first reation an therefore be irumvented. intermediate that requires subsequent strutural reorganization for the orret refolding

17 CHAPTER II Refolding of Urea-denatured Ovalbumin Comprises Non-Native Disulfide Isomers In former hapter, the author demonstrated the refolding pathway of ovalbumin (see Sheme 1 in hapter I). It shows the native disulfide in the aid/urea-denatured ovalbumin undergoes nonspeifi disulfide rearrangements in an initial burst intermediate and then is reovered during the subsequent slow refolding. Furthermore, the pathway shows that the non-native disulfide isomers possibly refold to native state via sulfhydryl/disulfide exhange reations. FIG. 6. Shemati view of ovalbumin. The figure is based on the X-ray rystallographi data of ovalbumin (26) and was drawn using the MolSript program ( 45). The numbered shaded spheres represent the sulfur atoms:l Cysll 2 ' ' ' Cys3; 5, Cys367; 6, Cys382. The sulfur atoms 3 (Cys73) and 4 (Cys12) form the native disulfide bond. In this hapter, the author investigates whether or not the urea-denatured ovalbumin omprising non-native disulfide isomers, D[non-N], an refold into N[3-4] with the orret disulfide. As desribed in a previous report (2), fifteen non-native disulfide isomers are produed when disulfidebonded ovalbumin is denatured at high onentrations of urea at near neutral ph. Aording to the X-ray rystallographi data (26), however, only the native disulfide Cys73-Cys12 an adopt the native onformation (Fig. 6). The refolding proess of ovalbumin denatured in 9 M urea at ph 8.2 was investigated by using several different strutural approahes. The author reports here that most, if not all, of the D[non-N] speies an orretly refold into the native form via disulfide rearrangements aording to an extended version of Sheme 1. Materials EXPERIMENTAL PROCEDURES At-ovalbumin was purified as desrt'bed ~ In 1.rmer hapter. Diphenylarbamyl hloride-treated trypsin (Type XI) and hymotrypsin (Type II) were purhased from Sigma. Ahromobater protease I (E.C ) was obtained from Wako Pure Chemial Industries. Denaturation and Refolding of Ovalbumin Denatured ovalbumin was prepared by inubating the native, disulfidebonded protein at 1. mg/ml, 37oC for 3 min in buffer A (5 mm Tris HCl buffer, ph 8.2, 1. mm Na-EDTA) ontaining 9 M urea. Refolding was initiated at 25oC by 2-fold dilution of the denatured protein with

18 buffer A, giving a final urea onentration as low as.45 M. The proteins were allowed to refold at the same temperature, and then analyzed for trypsin resistane, intrinsi tryptophan fluoresene and CD spetrum. An aid-quenhed equilibrium intermediate was produed by 2-fold dilution of the urea-denatured protein with 5 mm K-phosphate, ph 2.2 ontaining 1. mm Na-EDTA. The buffers were degassed at redued pressure and equilibrated under an N 2 atmosphere prior to the refolding experiments. Measurement of Intrinsi Tryptophan Fluoresene and CD Spetrum The fluoresene spetrum of ovalbumin was measured with a Hitahi fluoresene spetrophotometer (Model F-3). The intrinsi tryptophan residues in ovalbumin were exited at 295 nm and the time ourse of fluoresene intensity hange was monitored at 338 nm emission. The far- UV CD spetrum of ovalbumin was reorded with a spetropolarimeter (JASCO, J-72). All measurements were arried out at a onstant temperature of 25 C. The manner of measurements and so forth were arried out as desribed in former hapter. For rapid mixing experiments, a stopped-flow rapid kinetis aessory(applied Photophysis, RX.1) attahed to the same spetropolarimeter was employed and hanges in the CD elliptiity of the urea-denatured ovalbumin at 23 nm were reorded at 25 C after 1-fold dilution with buffer A. The dead time for mixing, determined by using the reation between 2,6-dihlorophenolindophenol and L-asorbate (25), was 2 ms. Trypsin-resistane Assay Refolded ovalbumin with the native onformation is onsidered to be a trypsin resistant speies as desribed (21). At various refolding times, the protein was mixed with.1 volume of different onentrations (1.25 or 12.5 mglml) of trypsin and digested at 25 C for 1 min. The digestion was terminated by addition of soybean trypsin inhibitor. The proteins were eletrophoresed on a sodium dodeyl sulfate polyarylamide gel (1% polyarylamide/.27% bisarylamide) aording to the standard method of Laemmli ( 41 ), and then stained with Coomassie brilliant blue R-25. The amount of trypsin-resistant ovalbumin was determined from the band intensity, whih was measured with a densitometer (Shimadzu, CS-9). Analyses for Disulfide-involved Half-ystines At various refolding times, sulfbydryl/disulfide exhanges were quenhed by mixing the protein samples with.24 volume of 2 M HCI. Disulfide-involved half-ystines were determined by alkylation with a fluoresent reagent IAEDANS, and subsequent peptide mapping proedure as desribed elsewhere (2). Determination of Cysteine Sulfhydryls by Amino Aid Analysis The quenhing of sulfbydryl/disulfide exhanges in the refolding protein by aid, alkylation of free ysteine sulfbydryls with iodoaetamide, and protein preipitation in a old aetone-hci solution were arried out in the same way as desribed in former. The alkylated protein was hydrolyzed

19 in 6 N HCl ontaining.1 % (v/v) phenol in the gas phase at 11 C for 2 whih the denatured protein is plaed in a refolding buffer of near-neutral h under vauum. The arboxymethyl ysteine was determined with an ph. A partially folded intermediate state, IN, has been shown to be amino aid analyzer (Hitahi, L-85A). formed in an initial burst phase, as deteted by far-uv CD and intrinsi Differential Sanning Calorimetry tryptophan fluoresene spetra, in either the disulfide-bonded or disulfideredued ondition. The partially folded equilibrium intermediate with The denatured protein was refolded for 2 h, onentrated about 15-fold using a onentrator (Amion, Centriprep-1), and passed through a Sephadex olumn (Pharmaia Bioteh, NAP-1) equilibrated with 1 mm Na-phosphate buffer, ph 6.. Overall reoveries from the original denatured protein were about 7 %. The refolded protein and native strutural harateristis equivalent to those of IN is formed upon the dilution of the aid/urea-denatured ovalbumin with an aidi buffer, ph 2.2. In this hapter, the author examined similarly whether or not the initial burst intermediate is also formed during the refolding from the ureadenatured ovalbumin at near neutral ph, where different disulfide isomers protein ontrol were analyzed with a differential sanning alorimeter should be present (2). As shown in Fig. 7, the refolding proteins from (Miro Cal, MCS-DSC). The protein onentration was.4 mg/ml in 1 mm Na-phosphate buffer, ph 6.. The rate of temperature hange was 1 K min- 1. RESULTS Folding Intermediate Deteted by Far-UV CD and Intrinsi Tryptophan Fluoresene Spetra In hapter I, the refolding mehanism of ovalbumin in either a disulfidebonded or a disulfide-redued ondition was investigated using a starting denatured protein sample produed by protein inubation in 9 M urea at ph 2.2, where possible sulfhydryl/disulfide exhange reations are almost ompletely bloked; the refolding was initiated by a ph-jump proedure in the urea-denatured state showed at the early stage of 1 s an intermediate CD spetrum that had 6% of the absolute elliptiity at 222 nm of the native form. When the time ourse of CD elliptiity hange was monitored using a rapid mixing apparatus, the obtained data were onsistent with ompletion of the formation of the intermediate from the urea-denatured state within the mixing dead time of 2 ms (Fig. 7, inset). Ovalbumin ontains three tryptophan residues, W148 in helix F, W184 as the nearest neighbor residue of the C-terminus of strand 3A and W267 in helix H (26). As shown in Fig. 8, the fluoresene emission spetrum of the native form had an emission maximum at 338 nm. The ureadenatured protein showed a typial red-shifted spetrum of an unfolded protein: the emission maximum was shifted to a longer wavelength of 352 nm, and the fluoresene intensity was dereased to 56 % of the native 3 3 1

20 form. When the urea-denatured ovalbumin was refolded, the protein showed at an early refolding time of 1 s a fluoresene spetrum that had a peak at the same wavelength, but with muh dereased intensity as ompared with the spetrum of native ovalbumin. - T'"" I N E () ' C? -2 x -4 ~ -6-8 Wavelength(nm).5 Time(s) FIG. 7. Far-UV CD spetra of various states of ovalbumin. The far-uv CD spetra of native ovalbumin (thik solid line), the protein denatured in 9 M urea (thik broken line), and the proteins refolded for 3 min and 2 h (the upper and lower thin solid lines, respetively) were reorded at 25 C as desribed in the text. The equilibrium intermediate state was produed by 2-fold dilution of the ureadenatured protein with 5 mm K-phosphate buffer, ph 2.2 ontaining 1. mm Na EDTA and the CD spetrum was reorded in the same way (thin dotted line). For the CD spetra for early refolding intermediates, the time ourse of hanges in CD elliptiities at different wavelength were reorded during the refolding and the values at the refolding time of 1 s were plotted as a funtion of wavelength (open irles with thin solid line). In the inset, the urea-denatured protein was refolded at ph 8.2 and the hanges in CD elliptiities at 23 nm were reorded for one seond using a stoppedflow instrument as desribed in the text. D and N shown by arrows represent the CD elliptiities at 23 nm of the urea denatured and native proteins, respetively. u u en g 4 ~ LL 2, _, I I Wavelength(nm) FIG. 8. Fluoresene emission spetra. 42 The tryptophan residues in native ovalbumin (thik solid line) and in the protein denatured in 9 M urea (thik broken line) were exited at 295 nm, and the emission spetrawere reorded at 25 C.The intrinsi tryptophan fluoresene spetra of the protein refolded for 3 min and 2 h (lower and upper thin solid lines, respetively) were reorded in the same way. The thin dotted line represents the fluoresene spetrum for the equilibrium intermediate state that was produed by 2-fold dilution of the urea-denatured protein with 5 mm K-phosphate buffer, ph 2.2 ontaining 1. mm Na-EDTA. For the fluoresene spetra at an early refolding time, the time ourse of fluoresene emission hanges were reorded during the refolding at different emission wavelengths and the values at refolding time 1 s were plotted as a funtion of emission wavelength (open irles with thin solid line). The fluoresene intensity is shown in arbitrary units. Figs. 7 and 8 also show that after the initial burst phase, the absolute CD elliptiity and fluoresene intensity then inreased slowly with inreasing time of refolding. The protein refolded for a prolonged time of 2 h

21 showed 9% of the absolute CD elliptiity at 222 nm and 83% of the at a trypsin onentration as low as 12.5 mg!ml. intrinsi fluoresene intensity at 338 nm of the native protein. The data Likewise, the aidquenhed intermediate that had strutural harateristis equivalent to the from the two onformational analyses were therefore onsistent with the initial burst refolding intermediate was almost ompletely degraded at the formation of an initial burst intermediate having 6 % of the absolute CD trypsin onentration of 12.5 mg!ml. This protease-sensitive nature elliptiity at 222 nm and 57% of the fluoresene intensity at 338 nm of the native form, during the refolding proess. The partially folded intermediate showing the same far-uv CD spetrum as the intermediate formed at ph 8.2 was also formed by dilution of the urea-denatured protein with an aidi buffer, ph 2.2 (Fig. 7). Under the should not be aounted for by the ph-jump proedure for the analysis of the aid-quenhed intermediate, sine native ovalbumin that had been preinubated at ph 2.2 showed protease resistane at 125 mg!ml of trypsin. The data in Fig. 9 therefore imply that the native protein, but not the ureadenatured or initial burst intermediate, is deteted as the resistant moleule same onditions, the fluoresene spetrum of ovalbumin showed the maximum at 338 nm that is onsistent with the peak wavelength for the at a protease onentration higher than 12.5 mg!ml. The time ourse for the refolding was examined by means of the trypsin intermediate formed at ph 8.2 (Fig. 8). The dereased fluoresene resistane assay and the data were ompared with the time ourses, after intensity for the aid-quenhed intermediate an be aounted for by the initial burst phase, obtained by the far- UV CD and intrinsi solvent effets owing to aid (23,4). These spetral profiles were almost fluoresene analyses. Fig. 1 learly demonstrates that the time ourse exatly the same as those of the previously observed intermediates formed for the refolding was almost exatly the same for the three onformational from the aid/urea-denatured protein ( 4). probes. The refolding data were fitted well to a biphasi rate equation (the sum of two exponentials). Suh biphasi kinetis is onsistent with Time Course for the Refolding after the Initial Burst Phase In a previous report (21 ), we have shown that a trypsin resistane assay the involvement of disulfide rearrangements during the refolding (see DISCUSSION). is a sensitive probe for the analysis of the native onformation of ovalbumin. The trypsin resistane of different onformational states of ovalbumin was examined in more detail. As shown in Fig. 9, native ovalbumin was resistant to trypsin at protease onentrations up to 125 mg!ml. Almost all of the urea-denatured protein, however, was degraded

22 en (1) (.) 8 (1) a. 6 (J) ts... en en 4 (1) ~ I en 2 a. ~ J Trypsin onentration (,_,._g/ml) FIG. 9. Trypsin resistane of various states of ovalbumin. Native ovalbumin dissolved at 1. mg/ml in buffer A (open irles) or in 5 mm K phosphate buffer, ph 2.2 ontaining 1. mm Na-EDTA (open triangles), or the protein denatured in buffer A ontaining 9 M urea (losed irles) was diluted 2-fold with buffer A ontaining different onentrations of trypsin to give the final protease onentrations shown on the absissa. After 1 min of inubation at 25 C, proteolysis was terminated by addition of soybean trypsin inhibitor. The samples were analyzed for sodium dodeyl sulfate polyarylamide gel eletrophoresis, and the amounts of intat ovalbumin were estimated as desribed in the text. The equilibrium intermediate (losed triangles) that had been produed by 2-fold dilution of the urea-denatured protein with 5 mm K-phosphate buffer, ph 2.2 ontaining 1. mm Na-EDTA was also analyzed in the same way. The ordinate represents the amounts of the intat proteins at various protease onentrations, expressed as perentages of the values obtained without added protease. ::: z LL E.8 ~ LL.> (5 z ~ Refolding Time (min) FIG.lO. Time ourse for refolding after the initial burst phase. The urea-denatured protein was refolded at ph 8.2, 25 C, and the time-dependent onformational regain was monitored in terms of the CD elliptiity at 222 nm (losed irles), the intrinsi tryptophan fluoresene at 338 nm (open triangles) and the trypsin resistane (open irles) as desribed in the text. The ordinate shown by FN(t) represents the fration of the native form at the refolding time of t, alulated by using the equation: FN(t) = (Xo - Xr) I (Xo - XN), where Xo and X 1 are the initial values and the values at the refolding times of t, respetively. For the CD and fluoresene analyses, the values at 6 s refolding were taken as Xa. XN is the final value of the refolding. The values at the refolding time of 2 h were employed as XN; the XN values were 9, 83, and 8 % of the native values for the CD elliptiity, tryptophan fluoresene, and trypsin resistane analyses, respetively. The solid urves represent nonlinear leastsquares fits of the experimental data to a biphasi rate equation: FN(t) = A 1 + A 2 e-k 1 + A 3 e-k 1 Obtained onstants were: 1.8 for A 1, -.67 for A 2, -.34 for A 3,.831 for k 1, and.261 for k

23 Sulfhydryl/disulfide Exhanges during Refolding The preeding data demonstrate the involvement of an initial burst refold through intrahain sulfuydryl/disulfide exhange reations into the native disulfide form. intermediate in the refolding proess of the urea-denatured ovalbumin. The author's previous study has demonstrated that ovalbumin undergoes, in the initial burst intermediate, disulfide rearrangements via intrahain sulfuydryl/disulfide exhange reations (see Sheme 1 in hapter 1). These data suggest that non-native disulfide isomers inluded in the ureadenatured ovalbumin are refolded into the native form with the orret disulfide bond through disulfide rearrangements. To investigate this possibility, the author determined the disulfideinvolved half-ystines by means of the peptide mapping proedure at various refolding times. As shown in Fig. 11, any of the six ysteine residues was found to be involved in a disulfide bond at the refolding time. Suh a random distribution in the denatured protein rearranged in a slow reation to a less random state. For Cys73 and Cys12 that form the native disulfide, the disulfide-involved amounts were only 2% at refolding time, but they inreased with time of refolding. More than 7% of these ysteines partiipated in disulfides at a prolonged refolding time of 2 h. In ontrast, for the other four ysteines the disulfide-involved amounts dereased with time of refolding. During the refolding, the number of 1. +J C/) >-.8 () I " (/) >.6 ~ > u.. I 4. ".4 ~ '+= :::J. C/) E '+- '+-.2 o.. +J a: Refolding Time (min) FIG. 11. Disulfide rearrangements during refolding. 2. :::J At various times of refolding from the urea-denatured state, disulfide-involved ysteines for Cysll (open squares), Cys3 (losed squares), Cys73 (losed irles), Cys12 (open irles), Cys367 (losed triangles), and Cys382 (open triangles) were determined using a peptide mapping analysis as desribed in the text. The number of free ysteine sulfhydryls (ross) was determined by amino aid analysis. z free ysteine sulfbydryls was almost onstant; the values were from 3. 7 to 3.8, being essentially onsistent with the number of free ysteine sulfbydryls in native ovalbumin. These data are onsistent with the view that most, if not all, of the denatured non-native disulfide isomers an

24 Analysis of the Integrity of the Refolding by Differential Sanning Calorimetry The preeding data show that most, if not all, of the ovalbumin moleules refold into the native state within 2 h inubation (Figs. 1 and 11 ). The integrity of the refolding was also investigated by an alternative method of differential sanning alorimetry. As demonstrated in Figure 12, the protein refolded for 2 h showed a major thermal transition peak at 77.6 C, although a minor peak with lower melting temperature was also deteted. The major transition temperature was almost exatly the same as the value for native ovalbumin (77. 7 C). DISCUSSION In hapter I, the author investigated the refolding mehanism of ovalbumin using the aid/urea-denatured protein (in 9 M urea, ph 2.2) as the starting protein sample, sine possible sulfbydryl/disulfide exhange reations in urea-denatured ovalbumin are almost ompletely bloked (5). Refolding was initiated by a ph-jump proedure in whih the aid/ureadenatured protein is plaed in a refolding buffer of near-neutral ph (ph 8.2). It has been demonstrated using this refolding system that most of the denatured ovalbumin moleules an orretly refold via non-produtive disulfide rearrangements in an initial burst intermediate IN (see Sheme 1 in - 4 E -(1j (.).Y.... a. 6 2 hapter I ). In this hapter, ovalbumin was denatured at ph 8.2 in 9 M urea as the starting protein sample and then allowed to refold at the same ph value. Sine the isoeletri point of A 1 -ovalbumin is 4.58 ( 42), the eletrostati interations should be quite different in the urea-denatured onditions at the two different ph values. The author have, however, observed that ovalbumin is in a random oil state in the presene of a high onentration of urea either at ph 8.2 (2) or at ph 2.2 (Tatsumi, E. and Hirose, M., unpublished observation). This indiates that both systems Temperature ("C) FIG. 12. Differential sanning alorimetry analysis of the refolded ovalbumin. Ovalbumin refolded for 2 h and native protein as a ontrol were analyzed by differential sanning alorimetry in 1 mm Na-phosphate buffer, ph 6. with a temperature sanning rate of 1 K min- 1 Endothermi transition profiles for the native protein (N) and for the refolded protein (R) are arbitrarily shifted on the ordinate sale for larity. allow examination of the refolding proess from random oil state to native state. The major differene in the two refolding systems is in the disulfide strutures of the urea-denatured proteins: in a high onentration of urea at a near-neutral ph, but not at a strongly aidi ph, ovalbumin has been shown to undergo extensive disulfide rearrangements generating many disulfide isomers inluding the native disulfide isomer D[3-4] and 4 4 1

25 mispaired disulfide isomers D[non-N] that all ontain one disulfide and N[3-4]. Through disulfide rearrangements, IN[3-4] undergoes reversible four sulfhydryls in the moleule (2). The results of optial and trypsin-resistane analyses in the present report demonstrate that most, if not all, of the urea-denatured D[3-4] and interonversion with IN[non-N], and D[3-4] with D[non-N]. The data for the onformational regain after the initial burst phase fitted well to an equation onsisting of the sum of two exponentials (see the D[ non-n] an refold into the native state (Fig. 1). In addition, the legend of Fig. 1). The apparent rate onstants obtained by the data differential sanning alorimetry analysis revealed almost exatly the same denaturation temperature for the refolded and native proteins, although a fitting analysis (.831 for k 1, and.261 for k 2 ) should inlude both the first -order rate onstants for the disulfide rearrangements and for the minor shoulder peak with a lower denaturation temperature was deteted folding from IN to N[3-4] ( 4). Although, beause of the lak for the for the former protein form (Fig. 12). During the refolding, a partially initial values for D[3-4] and D[ non-n], the rate onstants for disulfide folded intermediate state was formed in the initial burst phase (Figs. 2 and 3); the far-uv CD and intrinsi tryptophan fluoresene spetra were almost exatly the same as those of the early intermediate state formed rearrangement and folding reations ould not be determined using a more sophistiated rate equation ( 4), the data in Fig. 1 learly demonstrate that the time ourse for the onformational regain followed biphasi kinetis. from the aid/urea-denatured protein ( 4). After the initial burst phase, As a mehanism for biphasi refolding kinetis, the involvement of the regain of the native disulfide via disulfide rearrangements was observed during the refolding, in whih the number of free ysteine sulfhydryls was parallel pathway that is related to is-trans isomerization of proline residues has been demonstrated (28). Ovalbumin ontains 14 proline residues (18). almost onstant (Fig. 11). These data were onsistent with refolding of As shown in a previous report ( 4), however, the biphasi refolding ovalbumin aording to an extended version of Sheme 1: kinetis of disulfide-bonded ovalbumin an be aounted for by the D[non-N] IN [non-n] t~ t~ [3-4] ---+> IN[3-4] --~> N[3-4] (Sheme 2) involvement of disulfide rearrangements rather than by proline isomerization, sine the disulfide-redued ovalbumin refolds with simple monophasi kinetis. The author's attempts to separate different disulfide isomers by ion where IN[3-4] and IN[non-N] are the initial burst refolding intermediate with exhange or reversed phase HPLC have all been unsuessful, probably the native disulfide and with a mispaired disulfide, respetively; the former beause of the large size of ovalbumin. This has made it diffiult to is the ompetent intermediate for subsequent folding into the native form, determine in detail the pathway of the disulfide rearrangements during the

26 refolding. Sheme 2, however, indiates that a protein that ontains a ystine disulfide along with ysteine sulfuydryls in the moleule may generally be a useful model for the investigation of protein folding mehanisms. First, if an anaerobi ondition is employed, the numbers of intrahain disulfide and sulfuydryls are the same as those in the native protein during the refolding in whih disulfide rearrangements are inluded (Fig. 11 ). This indiates that non-native disulfide isomers an refold into the native disulfide form by intrahain sulfuydryl/disulfide exhange reations without the help of a atalyti reagent or of an enzyme. A major problem enountered in oxidative refolding studies, related to protein sulfuydryl aessibility to an oxidizing agent, an therefore be irumvented in the sulfuydryl!disulfide protein. Seond, the unfolded protein that is usually employed as the starting protein for subsequent refolding should onsist of a vast number of onformational isomers. The possibility that different subsets of the onformational isomers refold at different rates an not be ruled out. A disulfide isomer that is produed in a sulfuydryl/disulfide protein under denaturing onditions orresponds to a subset of onformational isomers; the onformational entropy of an isomer depends on the number of amino aid residues separating the two half-ystine residues (2, 43, 44). If the refolding mehanisms are ompared for different disulfide isomers, the refolding pathway may be diretly related to the free energies of the original denatured states. CHAPTER III Charaterization of Refolding Intermediate of Ovalbumin Using Non-Native Disulfide Isomers In hapter II, the author reported that urea-denatured ovalbumin omprising non-native disulfide isomers refold to native state and most, if not all, of the D[non-N] speies an orretly refold into the native form via disulfide rearrangements aording to Sheme 2. A lear refolding mehanism, however, seems diffiult to get by using the mixture of disulfide isomers with an unknown pair of disulfide as the starting sample. In a previous study, a preparation method for urea denatured ovalbumin with the only disulfide Cys 367- Cys 382 (express by D[S-6]) has been established by using a hemial modifiation tehnique ( 46). In this hapter, the author more learly investigates refolding from non-native disulfide speies by use of D [5-6]. While Sheme 2 in hapter II denotes that any disufide isomers of ovalbumin must undergo IN[3-4] via sulfuydryl/disulfide exhange reations to refold from urea-denatured state to native state. We also established the method for preparation of another hemial modified ovalbumin whih ontains one non-native disulfide Cys 367-Cys 382, three ysteine sulfuydryls (Cys 11, Cys 3 and Cys 12) and arboxymethylated Cys 73 (express by D[3CM/5-6]), whose disulfide annot rearrange to the native form Cys 73-Cys 12 on aount of the bloked Cys 73. D[3CM/5-6] annot refold to native state and stops refolding at IN state. Utilizing, the unique strutural situations of D[3CM/5-6], the author

27 onludes that non-native disulfide prevents IN from refolding to native Refolding was initiated at 25 o by 2-fold dilution of urea-denatured state, and haraterized whole onformation of IN as very ompat near the proteins with buffer A giving a final ph value of 8.2. The proteins were native state. allowed to refold at 25 o and were then analyzed by trypsin resistane, intrinsi tryptophan fluoresene and CD spetrum. The buffers were EXPERIMENTAL PROCEDURES Materials A 1 -ovalbumin was purified as desribed 1n former hapter. Diphenylarbamyl hloride-treated trypsin (Type XI) and hymotrypsin (Type II) were purhased from Sigma. Ahromobater protease I (E.C ) was obtained from Wako Pure Chemial Industries. degassed at redued pressure and equilibrated under N 2 atmosphere prior to the refolding. An equilibrium intermediate IA was produed by 2-fold dilution of urea-denatured proteins with 5 mm potassium phosphate buffer, ph 2.2, ontaining 1. mm Na-EDTA. For the experiments in the D[3CM/SH] and D[SH] ondition, buffer A was ontaining.5mm dithiothreitol. Denaturation and Refolding of Ovalbumin D[5-6] was prepared by hemial modifiation methods with 2,2' dipyridyl disulfide as desribed elsewhere ( 46). D[3CM/5-6] was prepared by the same method as preparation of D[5-6] exept for the subsequent treatment for reduing. Redued protein was inubating at 37 C for 1 min in buffer A (5 mm Tris-HCl buffer, ph 8.2, 1. mm Na-EDTA) ontaining 4 mm IAA in ase of D[3CM/5-6]. D[3CM/SH] was prepared by inubating D[3CM/5-6] at a 2. mg/ml, 37oC for 3 min in buffer B (9 M urea, 5 mm DTT, 5 mm Tris-HCl buffer, ph 8.2, 1. mm Na-EDTA). Buffer substituted 9 M urea-.25 M HCl ontaining 1. mm Na-EDTA for buffer B through a Sephadex olumn (NAP-25, Pharmaia Bioteh In.) after reduing treatment. D[SH] is the same as urea denatured proteins in SH ondition desribed in hapter I. Measurement of Intrinsi Tryptophan Fluoresene and CD Spetrum The fluoresene spetrum of ovalbumin was measured with a Hitahi fluoresene spetrophotometer (Model F-3). The intrinsi tryptophan residues in ovalbumin were exited at 295 nm and the time ourse of fluoresene intensity hange was monitored at 338 nm emission. The far-uv CD spetrum of ovalbumin was reorded with a spetropolarimeter (JASCO, J-72). All measurements were arried out at a onstant temperature of 25 C. The manner of measurements and so forth were arried out as desribed in hapter I. Stopped-flow Analysis For rapid mixing experiments, stopped-flow reation analyzer (Applied Photophysis Ltd. UK) was employed. Changes in the fluoresene

28 intensity at 338 nm of the urea-denatured ovalbumin exited at 295 nm were reorded at 25 C after 1-fold dilution with buffer A. The dead time for mixing was determined 4 ms by using the reation between 2,6- dihlorophenolindophenol and L-asorbate (25). Size Exlusion Chromatography Analysis Blue dextran, sodium azide, A 1 -ovalbumin and the following proteins were used for olumn alibration; bovine liver atalase ( naalai tesque ), rabbit musle latate dehydrogenase (Sigma type XI), human transferrin (Sigma grade II), bovine serum albumin (Sigma), bovine erythroyte Trypsin-resistane Assay Refolded ovalbumin with the native onformation is onsidered to be a trypsin resistant speies as desribed (21). All proedure and analysis for assay were arried out as the same manner desribed hapter II. Differential Sanning Calorimetry The denatured protein was refolded for 2 h and onentrated about 15- fold using a onentrator (Amion, Centriprep-1). The refolded protein and native protein ontrol were analyzed with a differential sanning alorimeter (Miro Cal, MCS-DSC). The protein onentration was.5mg/ml in buffer A. The rate of temperature hange was 1 K min- 1 arboni anhydrase (naalai tesque ). HPLC were performed with a Shimazu UV absorbane monitor SPD-2AS (set at 22 nm). Analytial gel filtration experiments were performed by use of TSKgel G3SW XL olumn (7.8 mm I.D. X 3 em). Column temperature was maintained at 25 C. The flow rate through the olumn was maintained at.5 ml/min. Column was equilibrated with the following elution buffer: buffer A; for native state samples, refolding samples and olumn alibration, 5 mm K phosphate buffer (ph2.2); for la samples, 9 M urea-.25 M HCl; for ureadenatured samples. Native state samples or refolding samples were prepared by 2-fold dilution of 1mg/ml native or urea-denatured protein with buffer A, respetively. Urea-denatured samples and la samples were prepared by 2-fold dilution of 1mg/ml urea-denatured protein with 9 Analyses for Disulfide-involved Half-ystines At various refolding times, sulfhydryl/disulfide exhanges were quenhed by mixing the protein samples with.24 volume of 2 M HCl. M urea-.25 M HCl or 5 mm K-phosphafe buffer, respetively. Proedure of HPLC analysis and alulation of stokes radius were pursuant as desribed ( 47, 48). Disulfide-involved half-ystines were determined by alkylation with a fluoresent reagent IAEDANS, and subsequent peptide mapping proedure as desribed elsewhere (2). ANS Binding Experiments ANS in the presene of ovalbumin in various states were exited at 35 nm and emission spetra were reorded at the wavelength range from

29 nm to 61 nm with a fluoresene spetrophotometer (Hitahi, F-3). All measurements were arried out at a onstant temperature of 25 C. Native state samples, refolding samples, urea-denatured samples and la samples were prepared as deried above. ANS spetra were reorded after 3 s adding.5 volume of 4.2 mm ANS to eah sample, exept for measurements in the 3 s-refolding sample. In ase of the 3 s refolding samples, urea-denatured protein were diluted with buffer A ontaining 21,._.,M ANS. The time ourse of fluoresene intensity hange was monitored at 47 nm emission. For the spetrum measurements at 3 s-refolding time, the time ourse of fluoresene intensity hanges was monitored at various emission wavelengths with exitation at 35 nm, and data at a refolding time of 3 s were plotted. The final onentration of protein and ANS were and 2,._.,M, respetively. to inspet for the effet of arboxymethylated Cys 73. As shown in Fig. 13, the fluoresene emission spetrum of the native form had an emission maximum at 338 nm. The urea-denatured protein showed a typial redshifted spetrum of an unfolded protein: the emission maximum was shifted to a longer wavelength of 352 nm, and the fluoresene intensity was dereased to 32% of the native form. When the urea-denatured ovalbumin was refolded, the protein showed at an early refolding time of 5 ms a fluoresene spetrum that had a peak at the same wavelength, but with muh dereased intensity as ompared with the spetrum of native ovalbumin. As shown in Fig. 14, the refolding proteins from the ureadenatured state showed at the early stage of 1 s an intermediate CD spetrum that had 6% of the absolute elliptiity at 222 nm of the native form. Figs. 13 and 14 also show that after the initial burst phase, the absolute CD elliptiity and fluoresene intensity then inreased slowly RESULTS Folding Intermediate Deteted by Intrinsi Tryptophan Fluoresene and Far-UV CD Spetra In former hapters, two types of partially folded intermediates IN and la have been shown to be formed in an initial burst phase, as deteted by far UV CD and intrinsi tryptophan fluoresene spetra, from D[3-4 ], D[SH] and mixture of disulfide isomers D[mix]. In this hapter, the author has examined about two types of non-native disulfide speies D[S-6] and D[3CM/5-6]. D[3CM/SH] whih was prepared by reduing the disulfide Cys 367-Cys382 of D[3CM/5-6] also examined and ompared with D[SH] with inreasing time of refolding. The data from the two onformational analyses were therefore onsistent with the formation of an initial burst intermediate having 6% of the absolute CD elliptiity at 222 nm and 58% of the fluoresene intensity at 338 nm of the native form, during the refolding proess. The partially folded intermediate showing the same far-uv CD spetrum as the intermediate formed at ph 8.2 was also formed by dilution of the urea-denatured protein with an aidi buffer, ph 2.2 (Fig. 14). Under the same onditions, the fluoresene spetrum of ovalbumin showed the maximum at 338 nm that is onsistent with the peak wavelength for the 5 5 1

30 intermediate formed at ph 8.2 (Fig. 13). These spetral profiles were almost exatly the same as those of the previously observed intermediates formed from the aid/urea-denatured protein ( 4). It onfirmed that similarly the initial burst intermediates are also formed during the refolding from D[S-6], D[3CM/5-6] or D[3CM/SH]. > -~ C/) +.J 1..8 (.) (.).6 C/) ~ ::J.4 LL >.2 ~ ~ N a: ~ I C\.1 C") E "' E (.) C)..._., "' I ~ X,...-,.... CD Wavelength (nm) Wavelength (nm) FIG.13. Fluoresene emission spetra of various states of ovalbumin. Tryptophan residues in various states of ovalbumin were exited at 295 nm, and the emission spetra were reorded at 25 o as desribed under "Experimental Proedures." N (thik lines) represents native ovalbumin. D is the urea-denatured protein as the following: D[5-6](open triangles with thin solid line), D[3CM/5-6](open squares with thin solid line), D[3CM/SH] (open irles with thin dotted line), D[SH] (open diamonds with thin dotted line). The equilibrium intermediate state IA was produed by 2-fold dilution of D with a non-denaturing buffer, ph 2.2. The intrinsi tryptophan fluoresene of the proteins refolded from D for 2h(2H) was reorded in the same way. For the fluoresene spetra at 5ms(5MS ), the time ourse of fluoresene hanges was reorded during refolding from D at different emission wavelengths by use of a stopped flow analyzer (see Stopped-flow Analysis in Experimental Proedures), and the values at the refolding time of 5ms were plotted as a funtion of emission wavelength. The fluoresene intensity is shown by an arbitrary unit. FIG.14. Far UV CD spetra. The far UV CD spetra in various states of ovalbumin were reorded at 25 o as desribed under "Experimental Proedures." The designations of N, D, IA and 2H for the different ovalbumin states are the same as in Fig.l3. For the CD spetra of refolding for los(los, losed symbols), the time ourse of hanges in CD elliptiities at different wavelengths were reorded during, and the values at the refolding time of 1 s were plotted as a funtion of wavelength. Experiments for Deteting of Burst Phase by Stopped-flow Analysis. Change of fluoresene intensity during the initial burst phase was measured by use of a stopped-flow reation analyzer. As shown in Fig. 15, the obtained data was onsistent with ompletion of the formation of

31 intermediate from eah urea-denatured isomer within the mixing dead time of 4 ms. These results indiate that the initial burst phase proeeds regardless of the type of disulfide. Time Course for the Refolding after the Initial Burst Phase The time ourse for the refolding after the initial burst phase was examined by means of the trypsin resistane assay, far-uv CD and intrinsi fluoresene analyses (Fig. 16). It was almost exatly the same for the E.4 o (") (")... ro.3.2 >.1... (J)....4 N[SH] () () (J) '- ::J LL D[3CM/SH] D[SH] Time (ms) FIG.lS. Stopped-flow analysis for the initial burst phase. All proteins were exited at 295 nm, and the hanges in fluoresene intensity at 338 nm were reorded for 1 ms using a stopped-flow analyzer as desribed under "Experimental Proedures". IN is the refolded protein from eah urea-denatured protein (D[3CM/5-6]; a, D[5-6]; b, D[3CM/SH];, D[SH]; d) at ph 8.2. N[3-4] and N[SH] are native proteins under the disulfide-bonded and redued onditions (see hapter 1). N[3CM/SH] was prepared by inubating 12mg/ml N[SH] with buffer A in the presene of 4mM IAA at 37 C for lomin (only Cys 73 is arboxymethylated ompletely under the onditions). three onformational probes on refolding from D[S-6], D[3CM/SH] and D[SH]. The refolding data of D[S-6] was fitted well to a biphasi rate equation (the sum of two exponentials). Suh biphasi kinetis are onsistent with the involvement of disulfide rearrangements during the refolding. D[3CM/SH] refolded with simple monophasi first-order kinetis similarly to D[SH]. No signifiant effets of arboxymethylated Cys 73 were observed in the kineti after the initial burst phase. There are the most important point to be notied in the data of D[3CM/5-6]. Trypsin resistane and CD elliptiity did not reover in that. These results indiate that D[3CM/5-6] annot make up its seondary struture after the initial burst phase and should not refold to the native state. The fluoresene intensity, however, partly reover in the data of D[3CM/5-6]. Although this data suggests that the intermediate IN might be more stable after the initial burst phase, the author onsiders different fator (see DISCUSSION)

32 Differential Sanning Calorimetry Analysis of Refolded Ovalbumin _.... z Ll... E.8 () ft (d) ll '- ~ Ll.. CD > :p tl z '+- :p (.) tl '- u...6 (b).4.2 Intrinsi tryptophan fluoresene and far- UV CD spetra showed that most, if not all, of ovalbumin moleules refold into the native state at 2 h inubation from D[5-6] and D[3CM/SH] (Figs. 13 and 14). The integrity of native refolding was investigated more rigorously by an alternative method of differential sanning alorimetry. Fig. 17 demonstrates that the protein refolded from D[5-6] at 2 h inubation underwent thermal transition with almost the same melting temperature as did the native protein ounterpart; the thermal denaturation temperatures were 77.7 o for the native protein and 77.2 C for the refolded protein from D[S-6]. No Refolding Time (min) differene was deteted in the melting temperature among the disulfide redued protein N[SH], N[3CM/SH], refolded protein from D[SH] and refolded protein from D[3CM/SH]. Theses results indiate that disulfide FIG.16. Time ourse for refolding after the initial burst phase. The urea-denatured proteins (D[3CM/5-6]; a and a', D[5-6]; b, D[3CM/SH];, D[SH]; d ) was refolded at ph 8.2, 25 C, and the time-dependent onformational regain was monitored in terms of the CD elliptiity at 222 nm (open triangles), the intrinsi tryptophan fluoresene at 338 nm (open irles) and the trypsin resistane (open squares) as desribed in the text. The ordinate shown by FN(t) represents the fration of the native form at the refolding time of t, alulated by using the equation: FN(t) = (Xo - X ) 1 I (Xo - XN), where Xo and X 1 are the initial values and the values at the refolding times oft, respetively. For the CD and fluoresene analyses, the values at 6 s refolding were taken as Xo. XN is the final value of the refolding. The values at the refolding time of 2 h were employed as XN; the XN values were 98, 95, and 95 % of the native values for the CD elliptiity, tryptophan fluoresene, and trypsin resistane analyses, respetively. The solid urves represent nonlinear least-squares fits of the experimental data to a monophasi rate equation: FN(t) = AI + A 2 e-k 1 (a, and d) or biphasi rate equation: FN(t) =AI+ A 2 e-k 1 + A 3 e-k 1 (a 'and b). Obtained onstants were shown at Table 3. redued ovalbumin, whether whose Cys 73 is arboxymethylated or not, an refold orretly into the same protein energy states as the native proteins. At the data of refolded protein from D[3CM/5-6], a peak was almost undetetable. This data onludes that refolded protein from D[3CM/5-6] dose not refold to the stable moleule like the native state and stops refolding at the unstable intermediate. A slight bulge in the data may be aount for the minor ontamination from D[3CM/SH] whih generates during the preparation of D[3CM/5-6] and is ontained 7% lower

33 Sulfhydryl/Disulfide Exhanges during Refolding The previous my studies ( 4, 49) have demonstrated that ovalbumin '\ E '\ tl 15 (.).::s:...._.., a. (.) (a) (b) () (d) FIG.17. Differential sanning alorimetry analysis of refolded ovalbumin. Refolded protein from ureadenatured state (D[S-6]; b, D[SH]; d, D[3CM/SH]; f, D[3CM/5-6]; g) at 2 h inubation and native protein (N[3-4]; a, N[SH];, N[3CM/SH]; e, these are the same as in Fig.15) ontrols were analyzed by differential sanning alorimetry in buffer A, with a temperature sanning rate of 1 K min- 1 Endothermi transition profiles are arbitrarily shifted on the ordinate sale for larity. undergoes, in the initial burst intermediate, disulfide rearrangements via intrahain sulfhydryl/disulfide exhange reations. The fat leads to the suggestion that urea-denatured ovalbumin with a non-native disulfide an refold into the native form with rearrangement of disulfide to the native pau. To investigate this possibility, the author determined the disulfideinvolved half-ystines during refolding from D[S-6] and D[3CM/5-6] by means of the peptide-mapping proedure at various refolding times. As shown in Fig. 18A, where expresses the data of refolding from D[S-6], only Cys 367 and Cys 382 were deteted as disulfide-involved ysteines at the refolding time. The disulfide-involved Cys 367 and Cys 382 dereased immediately after the beginning of refolding ; onomitantly at this stage, all of the other four ysteines Cys 11, Cys 3, Cys 73 and Cys 12 were 1 deteted as the disulfide-involved ysteines. The disulfide-involved Cys 73 and Cys 12, however, both inreased gradually and the amounts were about 8% at 2 h refolding. In ontrast, for the other four ysteines the 5 disulfide-involved amounts dereased with time of refolding. These data are onsistent with the view that most, if not all, of urea-denatured (g) ovalbumin with a non-native disulfide an refold to the native disulfide Temperature ( C) form by intrahain sulfhydryl/disulfide exhange reations without the help of a atalyti reagent or of an enzyme. Moreover, these results suggest that the refolding intermediate IN is so flexible as to rearrange its disulfide with easy beause the disulfide exhange reations, at the least, our twie to

34 hange from Cys 367-Cys 382 to Cys 73-Cys 12. form Cys 73-Cys 12. Intrahain sulfhydryl/disulfide exhange reations ontinued in this protein at 2 h beause various disulfide isomers had 1. "iii -(/) >- "".8 > (A) 1. (B) r.8 ~.6.6 I "" ~.4.4 en i:s.2 L ~.2 a: Refolding Time (min) FIG.18. Disulfide rearrangements during the refolding. D[ 5-6] (in panel A ) and D[3CM/5-6] (in panel B ) were refolded at ph 8.2, 25 o. At various refolding times, the disulfide-involved ysteines for Cys 11 (open squares), Cys 3 (losed squares), Cys 73 (losed irles), Cys 12 (open irles), Cys 367 (open triangles), and Cys 382 (losed triangles) were determined using a peptide mapping analysis (2). The data are the averages for dupliate determinations. The solid urves represent linear or nonlinear least-squares fits of the experimental data: f(t) = A1 + A2t (Cys 73 in panel B), f(t) = A 1 + A 2 e-k (Cys 11 in panel B), f(t) = A 1 + Az e-k + A3 e-k (Cys 73 in panel A, Cys 3 in panel B and Cys12, Cys 367,Cys 382 in both panels). Obtained onstants were shown at Table 3. The solid urves of Cys 11 and Cys 3 in panel A were obtained by urves smoothing between the data. The data in Fig. 18B, where expresses the data of refolding from D[3CM/5-6], also showed only Cys 367 and Cys 382 were deteted as disulfide-involved ysteines at the refolding time and dereased after the beginning of refolding onomitantly inreasing of Cys 11, Cys 3 and Cys 12. Cys 73, of ourse, did not inreased during refolding beause of bloked sulfhydryl; as a result, the disulfide did not exhange to native ome to equilibrium as a mixture. The amounts of disulfide-involved Cys 11, Cys 3 and 12 were about 47%, and Cys 367 and Cys 382 were about 27% at 2 h refolding respetively. These data indiate that ovalbumin whose disulfide annot exhange to native form does not misfold to a partiular non-native disulfide moleule but stops a flexible intermediate with sulfhydryl/disulfide exhange reations after the initial burst phase. Furthermore, the amount of disulfide-involved Cys 382 dereased more rapidly than Cys 367, and rapid inreasing of Cys 11 ourred onomitant! y. The data suggest that onformation of the flexible intermediate rna y be ompat like the native form. The distane of Cys 11-Cys 367 is muh further than that of Cys 367-Cys 382 on primary struture but is the most approximate on tertiary struture of native ovalbumin expet for Cys 73-Cys 12 (Fig. 6 in hapter II). Size Exlusion Chromatograhy Analysis in Various State of Ovalbumin The author experimented size exlusion hromatograhy analysis by taking advantage of the refolded protein from D[3CM/5-6] as the initial intermediate IN and the diluted urea-denatured protein with aidi buffer as the stable intermediate la. As shown Table 2, the disulfide type of moleule hardly affeted stokes radius in the native ( A), la ( A) or urea-denatured ( A) states. Refolded protein from D[3CM/5-6] was ompat ( A) near the native protein rather than 6 6 1

35 the another intermediate la or extended urea-denatured protein at any refolding time. distinguishable from another intermediate la onformationally. ANS binding analysis more learly indiated the distintion between two intermediates. Fig. 19 shows the emission spetra of ANS in the presene TABLE2 Stokes radius of ovalbumin in various states. Stokes radius was alulated by use of size exlusion hromatography analysis as desribed under "Experimental Proedures". Native protein N[3-4], N[SH] and N[3CM/SH] are the same as in Fig. 15. Samples Stokes radius (A) N[3-4] N[SH] 3.3±.6 3.7±.12 of ovalbumin in various states. In the native and urea-denatured state, ovalbumin showed no detetable ANS binding. In ontrast, greatly inreased fluoresene emission with a peak at 472nm was observed in the presene of intermediate la with any disulfide. The protein refolded from urea-denatured state by diluting with buffer A showed low level of ANS binding even 3 s-refolding. The dereasing of ANS binding was, N[3CM/SH] Refolding from D[3CM/5-6] for 16min Refolding from D[3CM/5-6] for 12min Refolding from D[3CM/5-6] for 2h IA[3-4] IA[SH] IA[3CM/SH] la[ 5-6] IA[3CM/5-6] D[3-4] D[SH] D[3CM/SH] D[S-6] D[3CM/5-6] 31.± ± ± ± ± ± ± ± ± ± ± ± ± ±.19 onomitantly refolding, deteted and onsistent with time ourse for the refolding after the initial burst phase (Figs. 16, 2 and Table 3); refolded protein from D[3CM/5-6] kept even level of ANS binding for 2 h. These results indiate that the intermediate la has exposed hydrophobi ore, and the intermediate IN has losed onformation. ANS Binding Analysis The preeding study has demonstrated that the initial intermediate IN is, in spite of flexible moleule, very ompat near the native form and learly

36 ~ en ~ 8 +-' 6 u u en 4 '- ::J LL Wavelength (nm) FIG.19. ANS binding to various states ovalbumin. ANS in the presene of ovalbumin in various states were exited at 35 nm and emission spetra were reorded. The fluoresene intensity was not detetable in the presene of any native samples or urea-denatured samples (shown by N, D with an arrow). la and 3S represent the spetra in the presene of la samples and 3 s refolding samples, respetively from D[5-6](open triangles with thin solid line), D[3CM/5-6](open squares with thin solid line), D[3CM/SH] (open irles with thin dotted line), D[SH] (open diamonds with thin dotted line). ANS spetra were reorded after 3 s adding.5 volume of 4.2 mm ANS to eah sample, exept for the measurements in 3 s-refolding sample. In ase of 3s-refolding sample, ureadenatured protein were diluted with buffer A ontaining M ANS. The final onentration of ANS was 2 1-!M. >. 8.."t: en 6. Q.) Q.) (..) Q.) (..) en Q.) L- :::J LL ' ' I I ~.. (a) ~,_o () '~--- -e o -- J "()" g (d) <> <> Refolding Time (min) FIG.2. Time ourse of ANS binding during refolding. The urea-denatured protein (D[3CM/5-6]; a, D[5-6]; b, D[3CM/SH];, D[SH]; d) was refolded at ph 8.2, 25 C, and the time-dependent onformational regain was monitored in terms of ANS binding. ANS was exited at 35 nm, and fluoresene intensity was monitored at 47 nm emission after 3 s adding.5 volume of 4.2 mm ANS to refolding samples at various refolding time. Only in the measurements of 3 s-refolding sample, urea-denatured protein were diluted with buffer A ontaining M ANS. The final onentration of ANS was 2 1-1M. The solid urves represent linear or nonlinear least-squares fits of the experimental data: f(t) = A 1 + A 2 t (a), f(t) = A 1 + A 2 e-k 1 ( and d), f(t) = A 1 + A 2 e-k 1 + A 3 e-l 1 (b). Obtained onstants were shown at Table

37 TABLE3 The onstants obtained from least-squares fitting. Many kineti parameters were obtained from various time ourse experiments (A) and pepide mapping analysis (B) mentioned above (see Figs 16, 18 and 2). a FL, CD, TR and ANS represent the floresene, CD elliptiity, trypsin resistane and ANS binding analysis, respetively. bequations used fitting are as follows: Eq.I; f(t) =AI + A 2 e k l, Eq.II; A 1 + A 2 e kt + A 3 e k t' Eq.III; f(t) =AI + A 2 t. ysteine in B represents disulfideinvolved ysteine. (A) Refolding Probe a Equationb Amplitudes Rate onstants (min-1) from At Az A3 kl kz (5-6] FL+CO+TR Eq.II (5-6] ANS Eq.II (3CM/5-6] FL Eq.II (3CM/5-6] CO+TR Eq.I (3CM/5-6] ANS Eq.III O(SH] FL+CO+TR Eq.I O(SH] ANS Eq.l (3CM/SH] FL+CO+TR Eq.I (3CM/SH] ANS Eq.l (B) Refolding Cysteine Equationb Amplitudes Rate onstants (min-1) from At Az A3 k1 k2 (5-6] Cys 73 Eq.II (5-6] Cys 12 Eq.II (5-6] Cys 367 Eq.II (5-6] Cys 382 Eq.II (3CM/5-6] Cys 11 Eq.l (3CM/5-6] Cys 3 Eq.II (3CM/5-6] Cys 73 Eq.III [3CM/5-6] Cys 12 Eq.II (3CM/5-6] Cys 367 Eq.II [3CM/5-6] Cys 382 Eq.II DISCUSSION In former hapters, the author investigated the refolding mehanism of ovalbumin using the urea-denatured protein D[3-4 ], D[SH] or mixture of disulfide isomers D[ mix] as the starting protein samples. It has been demonstrated that most of the urea-denatured ovalbumin moleules an orretly refold via intrahain sulfbydryl/disulfide exhange reations in an initial burst intermediate IN (see Sheme 2 in hapter II). The present study has more learly demonstrated that urea-denatured ovalbumin with a non-native disulfide an orretly refold by use of D[S-6]. As shown Fig. 18A, it is obvious that the disulfide Cys 367-Cys 382 rearranges to naive disulfide Cys 73-Cys 12 via intrahain sulfbydryl/disulfide exhange reations in IN. Most of the protein refolded from D[5-6] is the same as the native protein in all aspets; spetrosopi analysis (Figs. 13 and 14), trypsin resistane (Fig. 16), melting temperature (Fig. 17) and its disulfide (Fig. 18A). These results lead to the onlusion that ovalbumin, if any, hardly misfolds and orretly refolds without the help of a atalyti reagent or of an enzyme on aount of the presene of the flexible refolding intermediate IN. IN is flexible enough to rearrange its disulfide with easy. The intermediate IN has haraterized by use of refolded protein from D[3CM/5-6] in the present study. Intrinsi tryptophan fluoresene and far-uv CD spetra showed that D[3CM/5-6] ould refold to an initial burst intermediate IN (Figs 13 and 14). Analysis by CD elliptiity, trypsin resistane and ANS binding showed that D[3CM/5-6] never reovered its seondary struture and refolded to native state after initial burst phase

38 (Figs 16 and 2). We an say that refolded protein from D[3CM/5-6] is the protein stops refolding in IN state. The intermediate IN an be haraterized that 6 % of seondary struture reovers to native (Fig. 14) and it has no trypsin resistane (Fig. 16), is unstable (Fig. 17) and is flexible enough to our intrahain sulfuydryl/disulfide exhange reations (Fig. 18). IN is, moreover, losed moleule (Fig. 19) and ompat near the native state (Table 2). However, inreasing of intrinsi tryptophan fluoresene intensity in the D[3CM/5-6] data after initial burst phase is inonsistent with being at a stop in IN state (Fig. 16). The only inonsistent data is possible to interpret that the different disulfide isomer of IN may emit the different fluoresene intensity. Inreasing the FN(t) probed by intrinsi tryptophan fluoresene in D[3CM/5-6] (Fig. 16) oinides with inreasing Cys 3 and Cys 12 in disulfide-involved ysteine data (Fig. 18B). The first rate onstants k 1 obtained by nonlinear least-squares were.1; intrinsi tryptophan fluoresene,.11; Cys 3,.12;Cys 12, respetively. Likewise, disulfide isomers generated from D[3CM/5-6] are exhangeable eah other and are equilibrium after 2 h inubation. Therefore, it seems reasonable to suppose that refolded protein from D[3CM/5-6] is regarded as the IN state protein omprising various disulfide isomers. Another intermediate la also has haraterized. It reovers 6 % of its seondary struture to native (Fig. 14) and has no trypsin resistane (hapter II) as the same IN. However, la is extend moleule exposed hydrophobi ore (Fig. 19 and Table 2) in ontrast to IN. The present study has demonstrated another important point. Ovalbumin with non-native disulfide never refolds to native state without rearranging its disulfide to naive form in IN. It is neessary for disulfidebonded ovalbumin to undergo IN[3-4] to refold to native state. While disulfide redued ovalbumin an refold to native protein. These fats lead to the onlusion that the native disulfide Cys 73-Cys 12 is not neessary to refold to native state but a non-native disulfide prevents the intermediate IN from refolding to native protein. It is probable that ovalbumin annot refold to native state without orret intra-moleular interation. The intermediate la, whih is extended moleule, is impossible to have orret intra-moleular interation beause of unusual harge of side hain at low ph. The intermediate IN, in spite of its ompat onformation near the native state, is also impossible to do beause of its non-native disulfide. Consequently, the intermediate la and IN[non-N] never refold to native state. For reasons mentioned above, the author presents Sheme 3 about refolding proess of ovalbumin from the urea-denatured state. Sine formation of the seondary struture in protein subjets to the loal side hain -side hain interations, methods for predition of seondary struture have often used (5-54). On the other hand, it has been evident and widely aepted that formation of the seondary struture subjets to not only the loal interations but also the non-loal interations from the speifi tertiary struture, reently (55-58). The speifi non-loal interations is greatly regarded at present protein studies; for example, a~ ~ transition related to prion diseases (59-63). The author onsiders that in ase of ovalbumin, the seondary struture refolding in initial burst phase

39 may subjet to the loal interations beause it an reover independent from ph or disulfide types. The remaining seondary struture refolding after initial burst phase may subjet to the speifi non-loal interations beause it annot reover without orret intra-moleular interation. The speifi non-loal interations may be so rigorously that annot aept only one mis-paired disulfide in ompat moleule near the native. D [non-n] Burst phase (Neutral ph) IN [non-n] (Sheme 3) SUMMARY It is generally diffiult to investigate the folding mehanism of relatively high moleular proteins with disulfide. In this thesis, refolding proess of ovalbumin from urea-denatured state was studied by use of analysis for intrahain disulfide rearrangements and disulfide isomers. Ovalbumin has unique strutural harateristis that is four ysteine sulfhydryls along with an intrahain disulfide in a single polypeptide hain. The harateristis enable to analyze the refolding pathways and intermediates with traing disulfide isomers without using oxidizing agents. D [3-4] Burst phase (Neutral ph) Burst phase (Aidi ph) la (3-4) la [non-n] N [3-4] In hapter I, refolding proess was investigated using disulfide-bonded and redued ovalbumin. Two types of refolding intermediates IN and la were deteted after initial burst phase that is omplete within the mixing dead time of 4ms. IN, whih generates by dilution with neutral ph buffer, refolds to native protein slowly whereas la, whih generates by dilution with aidi ph buffer, stops refolding. la, however, also refolds to native state in ase the equilibrium intermediate is plaed in a near neutral ph Shemati refolding of ovalbumin Urea-denatured ovalbumin D ([3-4]; with native disulfide, [non-n]; with non-native disulfide) refolds to intermediate IN or la whose seondary struture reover about 6~% to native protein (N[3-4]) through the initial burst phase (gray arrows). IN, wh~h generates by dilution with neutral ph buffer, is the ompat moleule ~ear the native protein whereas IM whih generates by dilution with aidi ph buffer, ls the. extended moleule exposed hydrophobi ore. IN is, however, unstable and so flextble as to rearrange its disulfide via sulfhydryl/disulfide exhange reations (white arrows; k+l and k are the rate onstants for onversion). IN[3-4] an refold to native protein with a f;~st -order rate (a b Jak arrow; k, is the rate onstant). IN [non-n] never ref~l ds to native protein without undergoing IN[3-4] via sulfhydryl/disulfide exhange reations. ondition. Intrahain sulfhydryl/disulfide exhange reations our in IN. Consequently, non-native disulfide speies generate during refolding no matter how refolding is started from only native-disulfide protein. The fat is opposed to the framework model that suggests about mehanism of protein struture onstrution. The author presents a shemati of refolding pathway of ovalbumin as Sheme 1 by means of kineti analysis. In hapter II, refolding proess was investigated using mixture of various disulfide isomers of urea-denatured ovalbumin. It was delared that most, 7 7 1

40 if not all, of the urea-denatured ovalbumin omprises non-native disulfide isomers an orretly refold into the native form via disulfide rearrangements aording to Sheme 2. In hapter III, refolding proess was investigated ustng hemial modified ovalbumin with a non-native disulfide Cys 367- Cys 382. Two refolding intermediates were haraterized that both their seondary struture reovered about 6% to native protein but their whole onformations were very different. IN is the ompat moleule near the native protein whereas IA is the extended moleule exposed hydrophobi ore. IN is, however, unstable and so flexible as to rearrange its disulfide via sulfhydryl/disulfide exhange reations. Consequently, non-native disulfide speies an orretly refold to native protein with disufide rearrangement to the native pair without the help of a atalyti reagent or of an enzyme. It was, therefore, delared that non-native disulfide speies stopped refolding in IN and never refolded to native protein without disufide rearrangement to the native pair. The author presented Sheme 3 about refolding proess of ovalbumin from urea-denatured state. ACKNOWLEDGEMENT The author would like to express her sinere gratitude to Dr. Masaaki Hirose Professor of the Researh Institute for Food Siene, Kyo to ' University, for his pertinent instrutions and generous support throughout the ourse of this researh. The author would like to express her heartfelt thanks to Dr. Shigeo Aibara, Assoiate Professor of the Researh Institute for Food Siene, Kyoto University, for his kind guidane and valuable omments. The author is deeply grateful to Dr. Nobuyuki Takahashi, Researh Assoiate of the Researh Institute for Food Siene, Kyoto University, for his kind help and tehnial advie to work. The author gratefully aknowledges to Dr. Honami Yamashita for her helpful suggestions and warm enouragement. Finally, the author wishes to express appreiation to the members of the Researh Institute for Food Siene for their helpful disussions and enouragement

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43 62. Dalal, S., Balasubramanian, S., and Regan, L. (1997) Folding and Design 2, R71-R Kuwata, K., Hosino, M., Era, S., Batt, S. E., and Goto, Y. (1998) J. Mol. Bioi. 283, PUBLICATIONS 1. Maki Onda, Eizo Tatsumi, Nobuyuki Takahashi and Masaaki Hirose: Refolding Proess of Ovalbumin from Urea-denature State. Evidene for the Involvement of Non Produtive Side Chain Interations in an Early Intermediate. J. Bioi. Chern., 272, (1997) 2. Maki Onda, Eizo Tatsumi, Nobuyuki Takahashi and Masaaki Hirose: Refolding of Urea-denature Ovalbumin that Comprises Non-native Disulfide Isomers. J. Biohem., 122, (1997) 3. Maki Onda and Masaaki Hirose: Refolding of Urea-denature Ovalbumin with a Non-native Disulfide Cys 367-Cys 382. Manusript in preparation