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A circular dichroism study of the cyanogen bromide fragments of soybean trypsin inhibitor (Kunitz)
327
Biochimica et Biophysica Acta, 532 (1978) 327--336 © Elsevier/North-HollandBiomedicalPress
BBA 37832 A CIRCULAR DICHROISM STUDY OF THE CYANOGEN BROMIDE FRAGMENTS OF SOYBEAN TRYPSIN INHIBITOR (KUNITZ)
CLAUDIO TONIOLO, GIAN MARIABONORA, CLAUDIO VITA and ANGELO FONTANA Institute of Organic Chemistry, Biopolymer Research Centre, C.N.R., University of Padova, 35100 Padova (Italy)
(Received June 3rd, 1977) (Revised manuscript received September 29th, 1977)
Summary Fragmentation of soybean trypsin inhibitor (Kunitz) at the level of Met-84 and Met-114 with cyanogen bromide and subsequent reduction of disulfide bridges, S-carboxymethylation and gel chromatography have been employed to obtain in pure form fragments 1--84, 85--114, 115--181, as well as fragment 1--114 with the internal peptide bond 84--85 cleaved. Conformational analysis using circular dichroism in the far-ultraviolet region has been carried out in aqueous solution on the isolated fragments, the native inhibitor, and its reduced S-carboxymethylated derivative. Whereas the native inhibitor does not appear to possess any regular structure, reduction of disulfide bridges and S-carboxymethylation leads to the appearance of some significant regular conformation. Fragments 1--84 and 1--114 are the most prone to assume a regular structure (essentially a ~-conformation), whereas fragments 85--114 and 115--181 are structureless. By using structure-promoting agents (trifluoroethanol and sodium dodecyl sulfate} it has been possible to induce significant levels of a-helix in all the compounds examined. The results obtained appear to support the hypothesis that partial cleavage of the polypeptide chain and reduction of disulfide bonds permit the formation of a more regular structure than exists in the native inhibitor.
Protein conformation is determined by a number of forces, which are often divided into short-, medium- and long-range interactions [1--3]. A well-established experimental approach for elucidating the nature and magnitude of such Abbrevlations: ABC, cyanogen bzomide fragment 1--114; AB-CM, S-carboxymethylated fragment 1--84; C-CM, S-carboxymethylated fragment 85--114; D, fragment 115--181; D-CM, S-carboxymethylated fragment 115--181; SDS, s o d i u m dodecyl sulfate; Hse, homoserine; Tris, tris(hydroxymethyl)aminomethane.
328
interactions is represented by conformational studies of peptide fragments of fully characterized proteins [4--14]. Generally, it has been shown that in aqueous solution peptide fragments contain less secondary structure than the corresponding sequences in the parent protein. The conclusion reached from studies of fragments of myoglobin [4--6], ribonuclease [7--9], nuclease [10-12], hemoglobin [13], and cytochrome C [14] is that in general long-range interactions increase the amount of regular conformation in proteins. In the present paper we report a far-ultraviolet circular dichroism study of the secondary structure of reduced and S-carboxymethylated cyanogen bromide fragments of soybean trypsin inhibitor (Kunitz). This protein was shown by X-ray crystallography to possess little regular structure, most of the polypeptide chain being involved in only approximate pleated-sheet ~-form and with no a-helix [15]. The results reported here indicate that partial peptide bond cleavage and reduction of disulfide bonds of the protein induce the tendency of part of the polypeptide chain to form more regular structure (mostly /3-structure) than exists in the native protein. Materials and Methods Soybean trypsin inhibitor (Kunitz), type I-S, was obtained from Sigma (St. Louis, Miss., U.S.A.). Cyanogen bromide, 2-mercaptoethanol, 2,2,2-trifluoroethanol, dithiothreitol, iodoacetic acid, Tris, and 98--100% formic acid were Fluka (Basel, Switzerland) products and were used without further purification. Urea (Erba, Milan, Italy) was recrystallized from 95% ethanol and only fresh solutions of the reagent were used. Sodium dodecyl sulfate (Fluka) was recrystallized from methanol/benzene. Diethylaminoethyl-cellulose (DE-52) was obtained from Whatman (Maidstone, U.K.) and Sephadex G-50 SF from Pharmacia (Uppsala, Sweden). All other reagents were analytical grade and used without further purification.
Purification of soybean trypsin inhibitor The commercial sample of the inhibitor (200 mg) was purified on a column (2 X 25 cm) of diethylaminoethyl-ceUulose (DE-52) equilibrated with 0.01 M ammonium acetate buffer, pH 6.5 [16]. The column was eluted with a linear gradient of the same buffer from 0.01 to 0.5 M, at a flow rate of 25 ml/h. Fractions of 6 ml were collected and the protein located in the effluent by absorption measurements at 280 nm. The fractions corresponding to the main peak of the protein were combined and then the protein recovered by lyophilization. The protein sample was rechromatographed on the same column [16], eluted in the same conditions, and again recovered by lyophilization. This sample was used in the present study for fragmentation with cyanogen bromide.
Cleavage with cyanogen bromide [17] Purified soybean trypsin inhibitor (100 mg) was reacted with cyanogen bromide (110 rag; approx. 50 equiv.) in 10 ml of 70% formic acid for 24 h in the dark. The reaction mixture was then diluted with water and evaporated in vacuo. The concentrated solution was then applied over a column (3.3 X 140 cm) of Sephadex G-50 Superfine equilibrated and eluted with acetic acid/ formic acid/water (40 : 10 : 50, v/v). The effluent was monitored by absorp-
329 tion measurements at 280 nm. Three peaks of peptide material were obtained, which were shown, by amino acid analysis, to correspond to uncleaved soybean trypsin inhibitor (~ 10%) and fragments ABC and D. The effluents corresponding to the two fragments were concentrated and rechromatographed on the same column. Purity of the peptides was tested by amino acid analysis. Reduction and S-carboxymethylation 1. Soybean trypsin inhibitor. To a solution of 20 mg of purified soybean trypsin inhibitor in 2 ml of 8 M urea in 0.1 M Tris pH 8.2, 3.1 mg of dithiothreitol were added and the pH adjusted to 9.5 with 0.1 M NaOH. The reaction mixture was left at room temperature in the dark for 4 h and then the pH was adjusted to 8.0 with 0.1 N HC1. Iodoacetic acid (15 mg) was dissolved in 0.5 ml of water and the pH adjusted to 8.0 with 0.1 M NaOH and then added to the solution of the reduced soybean trypsin inhibitor. The excess of alkylating agent was removed by the addition of 70 pl of mercaptoethanol and the pH was kept at 7.0 for 15 min. The reaction mixture was directly applied to a Sephadex G-25 Superfine column (2 X 50 cm) equilibrated with 10% acetic acid. The effluent was analyzed at 280 nm.The protein peak was collected and the reduced and carboxymethylated soybean trypsin inhibitor was recovered by lyophilization. 2. Fragment ABC. Reduction and carboxymethylation of fragment ABC were carried out as above. The reaction mixture was then applied to a Sephadex G-50 Superfine column (3.3 X 140 cm) equilibrated with acetic acid/formic acid/water (40 : 10 : 50, v/v). Fragments AB-CM and C-CM were completely separated from each other. The peaks of the fragments were preceded by trace amounts of uncleared fragment ABC. 3. Fragment D. Reduction and carboxymethylation of fragment D were carried out as above. Fragment D-CM was separated by gel filtration on a Sephadex G-25 Superfine column (2 X 50 cm) equilibrated with 10% acetic acid and recovered by lyophilization. Methods
Measurements of absorbance at single wavelengths were carried out with a Hitachi-Perkin-Elmer spectrophotometer, model 139. Continuous spectra were recorded with a Cary 15 spectrophotometer. Circular dichroism spectra were measured with a Cary dichrograph, model 61. Cylindrical fused quartz cells with 0.5- and 1-mm path length were used. The instrument was flushed with prepurified nitrogen before and during the experiments. In the spectra the mean residue ellipticity values [0 ] are reported. They are defined as follows: [0] = (O/lO)(M/lc), where 0 is the measured ellipticity in degrees, l is the path length of the solution in cm, c is the concentration in g/cm 3, and M is the mean residue molecular weight. The latter was taken as 111.1 for soybean trypsin inhibitor, 108.0 for fragment ABC, 112.2 for reduced and carboxymethylated soybean trypsin inhibitor, 109.2 for AB-CM, 107.3 for C-CM, 115.8 for D, and 117.4 for D-CM. The various right-handed s-helical contents of the reduced and S-carboxymethylated trypsin inhibitor
330 and peptide fragments were calculated using the relationship proposed by Greenfield and Fasman [18] on the basis of the ellipticity values at 208 nm. The concentrations of solution were determined by quantitative amino acid analysis of solutions of known absorption and then checked routinely by measurements of absorbance at 280 nm. The pH values were measured with a Metrohm pH meter, model E-510, equipped with a combined glass electrode. Results
Preparation and characterization of fragments The amino acid sequence of soybean trypsin inhibitor contains 181 amino acid residues, and in particular two disulfide bridges (Cys39-Cysa6 and Cys 13~Cys 14s) and two methionine residues (in positions 84 and 114) [19]. The peptide bond between Args3 and Ile 64 is split on interaction with trypsin, giving a modified soybean trypsin inhibitor with a cleaved peptide bond [17,20]. Cyanogen bromide cleavage [21] of the protein yields three fragments, namely 1--84, 85--114 and 115--181. Since fragments 1--84 and 85--114 are linked together via a disulfide bridge, gel filtration on a Sephadex G-50 SF column allows separation of sequences 1--114 (ABC) and 115--181 (D). Reduction and S-carboxymethylation of fragment ABC according to general procedure (ref. 22 and references therein) and passage of the reaction mixture on a Sephadex G-50 SF column allows complete separation of the S-carboxymethylated sequences 1--84 (AB-CM) and 85--114 (C-CM). Native soybean trypsin inhibitor and fragment D were also reduced and S-carboxymethylated, and the derivatives (reduced and carboxymethylated soybean trypsin inhibitor and D-CM, respectively) were purified by gel filtration. The general scheme of fragmentation of native soybean trypsin inhibitor is shown in Fig. 1. The nomenclature of the fragments used is similar to that proposed by Koide and Ikenaka [17]. The purity of soybean trypsin inhibitor, its fragments and their S-carboxymethylated derivatives was tested by amino acid analysis (Table I). Analyses were in good agreement with theory [19]. The low values of leucine residue found only in the intact inhibitor, fragment D, and their reduced and S-carboxymethylated derivatives confirm that some molecules of the inhibitor lack C-terminal leucine [17]. The low recovery on the analyzer of valine and isoleucine residues should be related particularly to the presence of Val-Val, ValIle and Ile-Ile sequences, which are known to be difficult to hydrolyze [17,19, 23]. The above hypothesis is in part confirmed by the observation that the values of isoleucine residue are low only in the peptides where Val-Ile and IleUe bonds occur (soybean trypsin inhibitor, its reduced and carboxymethylated derivative, and fragments ABC and AB-CM}.
Circular dichroism studies Effect of pH. The far-ultraviolet circular dichroism spectra of native soybean trypsin inhibitor and fragments ABC and D at pH 1.0, 7.0 and 11.0 are shown in Fig. 2. The curve recorded for native soybean trypsin inhibitor at pH 7.0, exhibiting a positive maximum at 226 nm and a negative maximum near 200 nm, is in agreement with those reported in the literature where they have been
331 1 Asp
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Fig, 1. The schematic representation of the f r a g m e n t a t i o n of s oybe a n t ryps i n inhibitor.
interpreted as indicative of an aperiodic conformation [24--32]. The conformation of native soybean trypsin inhibitor does not appear to be significantly affected by the pH of the solution (Fig. 2A) [25,26,30]. Fragment ABC in 20 mM phosphate buffer, pH 7.0, shows a circular dichro-
TABLE I AMINO ACID COMPOSITION OF SOYBEAN TRYPSIN INHIBITOR, ITS REDUCED AND CARBOXYM E T H Y L A T E D D E R I V A T I V E AND CYANOGEN BROMIDE FRAGMENTS ABC, AB-CM, C-CM, D AND D-CM Values are not corrected for losses during acid hydrolysis. Theoretical values s.re shown in parentheseL Amino acid
STI *
STI-CM *
ABC
AB-CM
C-CM
D
D-CM
Aspartic acid Threonine Serine Glutamic acid Prollne Glycine Alanine Half-cystine
26.8 (26) 7.1 (7) 10.8(11) 18,3 (18) 10.6(10) 15.6 (16) 8,5 (8) 4.3 ( 4 ) -12.4(14) 2.1 (2) 12.6 (14) 14.3(15) 3.9 (4) 8,6 (9) 10.1 (10) 2.3 (2) 8.8 (9)
26.2 (26) 6.6 (7) 10.2(11) 18.6 (18) 10.7(10) 16.4 (16) 7.7 (8)
13.0 (13) 6.0 (6) 7.2 (7) 9.9 (10) 8.5 (8) 12.0 (12) 7.0 (7)
10.5 (10) 4,8 (5) 5,9 (6) 6,6 (6) 5,4 ( 5 ) 9.2 (9) 4.9 (5)
3.1 (3) 1.0(1) 1.0(1) 4.2 (4)
13.2 (13) 1.3 (1) 3.6 (4) 8.1 (8)
13.1 (13) 0.9 (1) 3.7 (4) 8.3 (8)
CM-cysteine
Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine LyJ~ne Histidine Arginine
--
3.8 (4) 10.8(14) 1.9 (2) 12.7 (14) 14.3(15) 3.9 (4) 8.9 (9) 10.2(10) 2.0 (2) 9.3 (9)
2.0
(2)
-6.8 (8) . . 9.6 (11) 8.7 (9) 2.6 (3) 5.0 (5) 3.9 (4) 1.1 (1) 5.3 (5)
3.6(3)
2.3
(2)
2.1
(2)
3.0 (3) 2.0(2)
4.0 1.2
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4.3 1.2
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2.4
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(3) (6) (1) (4)
2.9 5.2 1.1 3.6
(3) (6) (1) (4)
(2) (1)
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5.7 1.2
(6) (1)
6.4 1.0
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7.8 6.9 2.7 4.7 2.2 1.0 4.8
.
* STI, s o y b e a n t r y p s i n i n h i b i t o r ; STI-CM, S - c a r b o x y m e t h y l a t e d s o y b e a n t r y p s i n inhibitor.
332
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Fig. 2. F a r - u l t r a v i o l e t c i r c u l a r d i c h r o i ~ n s p e c t r a o f s o y b e a n t r y p s i n i n h i b i t o r and its c y a n o g e n b r o m i d e fragments A B C and D at r o o m t e m p e r a t u r e at p H 1 . 0 . . . . , in 2 0 m M s o d i u m p h o s p h a t e buffer• p H 7.0 • and at pH 11.0 ...... . A, native protein; B, f r a g m e n t A B C ; C, f r a g m e n t D.
ism spectrum with a negative band centered at approx. 205 nm (the corresponding mean residue ellipticity values is --5500 _+300 degree • cm 2 . dmol-') and a prominent shoulder near 217 nm (Fig. 2B) [24]. The characteristics of the curve indicate the presence of some significant regular structure. The conformation of fragment ABC is affected by acid and alkali, since a strong negative Cotton effect is generated and centered at 210--215 nm at pH 1.0 and 11.0. Conversely, fragment D appears to possess little regular structure in neutral, acid or alkaline aqueous solution (Fig. 2C), since regularly increasing negative ellipticity is shown in the region 250--200 nm with maxima near 200 nm [24]. Figure 3 shows the CD curves recorded at pH 1.0, 7.0, and 11.0 of reduced and c a r b o x y m e t h y l a t e d soybean trypsin inhibitor, AB-CM, C-CM and D-CM. The circular dichroism spectrum of reduced and carboxymethylated soybean trypsin inhibitor (Fig. 3A) at pH 7.0 is characterized by a negative band centered near 206 nm with an ellipticity value of --6500 +-300 degree • cm 2 • dmo1-1 and a shoulder near 215 nm [30]. Fragment AB-CM in aqueous buffer, pH 7.0, shows a negative band centered at 214 nm with an ellipticity value of --4500 + 300 degree • cm 2 • dmol -~ (Fig. 3B). The presence of regular structure in both cases is evident and the characteristics of the circular dichroism pattern of fragment AB-CM could be related to the occurrence of a significant extent of ~-conformation. Changing the pH of the solution from neutral to acid or alkaline region appears to induce conformational transitions both in reduced and c a r b o x y m e t h y l a t e d inhibitor and AB-CM. At acid pH the content of ~ o n f o r m a t i o n in reduced and c a r b o x y m e t h y l a t e d inhibitor is increased, whereas alkaline pH is effective in destroying the periodic structures. Fragments C-CM and D-CM exhibit CD spectra in neutral as well in acidic or alkaline solution indicative of peptide chains in an essentially aperiodic form. The state of aggregation of the various fragments under the experimental conditions employed for the circular dichroism measurements may have an
333 0 I { -2
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Fig. 3. F a r - u l t r a v i o l e t c i r c u l a r d i c h r o i s m s p e c t r a of r e d u c e d a n d S - c a r b o x y m e t h y l a t e d i n h i b i t o r a n d fragm e n t s AB-CM0 C-CM a n d D-CM a t r o o m t e m p e r a t u r e at p H 1.0 ( . . . . ), in 20 m M s o d i u m phosphate b u f f e r , p H 7.0 ( ), a n d a t p H 1 1 . 0 ( . . . . . . ). A, r e d u c e d a n d c a r b o x y m e t h y l a t e d i n h i b i t o r ; B, fragm e n t AB-CM; C, f r a g m e n t C-CM; D, f r a g m e n t D-CM.
influence on the spectral properties observed. Altough no molecular weight estimations were carried out to verify conclusively this point, it has been shown that circular dichroism data are independent of peptide concentration within the limits of experimental error. In particular, concentration effects have been studied in more detail with fragments ABC and AB-CM, which appear to possess some structure in aqueous solution (Figs. 2 and 3). Presumably, at the low concentrations used in the circular dichroism experiments (approx. 0.1 mg/ ml) the peptides are mostly in the monomeric form.
Effect of trifluoroethanol and SDS Native soybean trypsin inhibitor, its reduced and S-carboxymethylated derivative and all the fragments herewith investigated give in 98% trifluoroethanol a family of circular dichroism curves resembling that of helical polypeptides, i.e., with two negative maxima near 222 and 208 nm [34]. In particular, in each case the band at 208 nm is about 10% deeper than that at 222 nm. The amount of a-helix generated by the haloalcohol was calculated from the relationship proposed by Greenfield and Fasman [18] which makes use of the ellipticity value at 208 nm. The resulting percentages are reported in Table II. Alkyl sulfates with a long hydrocarbon chain, like SDS, are also effective in inducing the formation of a-helix in soybean trypsin inhibitor [27--31]. The effect of SDS was examined at 2% concentration and at pH 2.2, where the interaction between the anionic detergent and the polypeptide chain is maxi-
334 T A B L E II P E R C E N T O F R I G H T - H A N D E D { x - H E L I C A L C O N F O R M A T I O N IN S O Y B E A N T R Y P S I N I N H I B I T O R , ITS REDUCED AND S-CARBOXYMETHYLATED DERIVATIVE AND CYANOGEN BROMIDE F R A G M E N T S A B C , A B - C M , B-CM, D A N D D-CM C a l c u l a t i o n s w e r e m a d e a c c o r d i n g t o G r e e n f i e l d a n d F a s m a n [ 1 8 ] u s i n g t h e e l l i p t i c i t y v a l u e a t 2 0 8 rim. Compound
98% trifluoroethanol *
2% S D S pH 2.2
Soybean t~ypsin inhibitor CM-derivative of the inhibitor ABC AB-CM C-CM D D-CM
57 57 47 45 14 52 28
25 21 21 20 10 17 11
* 9 8 % t r i f l u r o r o e t h a n o l in 2 0 m M s o d i u m p h o s p h a t e b u f f e r , p H 7.0.
mal [28]. Comparatively to trifluoroethanol lower percentages of a-helix are observed in all compounds examined (Table II). Discussion
Quantitation of secondary structure of proteins can be obtained from an analysis of their CD spectra in the far-ultraviolet region [18]. However, there are several limitations in all the proposed methods of calculation [35] and the estimates should often be regarded as only qualitative. Nevertheless, farultraviolet circular dichroism measurements are useful in detecting structural properties and conformationai transitions of polypeptide chains. The far-ultraviolet circular dichroism spectrum of soybean trypsin inhibitor is indicative of an aperiodic conformation (Fig. 2A) in agreement with its crystal structure determined by X-ray diffraction [15]. The protein appears in fact as a sphere of approx. 35 A in diameter, made by criss-crossing loops wrapped around a core of hydrophobic side chains, with no a-helix and only irregular ~-pleated-sheet structure. The basic observation made in the present work is that fragmentation of the peptide chain of soybean trypsin inhibitor as well as reduction of disulfide bonds favor the formation of a significant amount of regular secondary structure in part of the inhibitor chain which is absent in the native molecule. In fact, the far-ultraviolet circular dichroism patterns of ABC, reduced and carboxymethylated inhibitor, and AB-CM suggest the existence in aqueous solution of polypeptide chains containing structurally periodic segments. We have found that the pH of the solution induces conformational transitions in some of the examined species, at variance with native inhibitor whose conformation is practically unaffected on going from neutral to acid or alkaline media [25, 26,30]. Since the N-terminal fragment (ABC) of the inhibitor is prone to assume a regular structure, while the C-terminal fragments C-CM, D and D-CM are essentially structureless, we propose that also in the reduced and S-carboxymethylated protein the structured region of the chain would be the N-terminal
335
one. This finding could bear upon the possibility role of the amino end of the chain in initiating protein folding [3]. The haloalcohol trifluoroethanol and the detergent SDS induce the onset of a-helical conformation into a considerable extent in all the compounds examined, as indicated by the circular dichroism spectra with negative maxima near 222 and 208 nm. Although in a few cases some deviation was observed from the standard a-helix spectrum given, for example, by Greenfield and Fasman [18], the mean residue ellipticity values at 208 nm were used to calculate the percentages of a-helix attained in the presence of trifluoroethanol and SDS. The extent of a-helix of reduced and carboxymethylated inhibitor in trifluoroethanol is substantially higher than in the three reduced and S-carboxymethylated fragments taken together. This could be related to end-group effects produced by cleavage of the 84--85 and 114--115 peptide bonds by cyanogen bromide, and/or possibly to the occurrence of long-range interactions [3] stabilizing helical segments in reduced and carboxymethylated inhibitor. However, the interpretation of all the figures reported in Table II is not straightforward and it does not seem to us worth a detailed discussion. This study may contribute to the comprehension of the mechanism of folding of proteins. In considering the structure of a protein in aqueous solution, short-, medium- and long-range interactions along the peptide chain are considered as determining the specific folded structure of the molecule [3]. Usually, short- and medium-range interactions are thought as being the most important and these are employed to predict secondary structure of proteins. Sometimes unsuccessful or poor predictions of protein structures result from the application of oversimplified rules which do not take into account longrange interactions. Analogously, the extent of periodic structure in fragments of globular proteins has usually been found to be far below the expected values and also this change in conformation has been related to the occurrence of long-range interactions which increase the amount of regular structure of the native polypeptide chain. A possible, although not the only, explanation of the data on fragments of a protein with complete aperiodic structure discussed here is that long-range interactions, though in most cases enhancing the percentage of periodic conformation of a peptide molecule, in certain limited cases could prevent the chain from assuming its maximal degree of regular structure. The present results parallel those recently reported by White [26] who was able to demonstrate that reduction of disulfide bonds in lysozyme and subsequent S-alkylation favors the attainment of more ~-structure than exists in the native protein. Acknowledgments The authors wish-to thank Mr. M. Zambonin, Mr. S. Fioretto Da Rin and Mr. F. Miozzo for expert technical assistance. References 1 Tanaka, S. and Scheraga, H.A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3 8 0 2 - - 3 8 0 6 2 Wetlaufer, D.B. an d Ristow, S. (19"/3) Annu. Rev. Biochem. 42, 135--158
336 3 4 5 6 7 8 9 I0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Anfinsen, C.B. and Scheraga, H.A. (1975) Adv. Protein Chem. 29,205--300 Crumpton, M.J. and Small, Jr., P.A. (1967) J. Mol. Biol. 26,143--146 Epand, R.M. and Scheraga, H.A. (1968) Biochemistry 7, 2864--2872 Hermans, Jr., J. and Puett, D. (1971) Biopolymers 10, 895---914 Scatturin, A., Tamburro, A.M., Rocch/, R. and Scoffone, E. (1967) Chem. Commun. 1273--1274 Klee, W.A. (1968) Biochemistry 7, 2731--2736 Brown, J.E. and Klee, W.A. (1971) Biochemistry 10,470--476 Anfinsen, C.B. (1972) Biochem. J. 128, 737--749 Taniuchi, H. (1970) J. Biol. Chem. 245, 5459--5468 Taniuchi, H. and Anfinsen, C.B. (1971) J. Biol. Chem. 246, 2291--2301 Fronticelli Bucci, C. and Bucci, E. (1975) Biochemistry 14, 4451--4458 Toniolo0 C., Fontana, A. and Scoffone, E. (1975) Eur. J. Biochem. 50, 367--374 Sweet, R.M., WrighL H.T., Janin, J., Chothia, C,H. and Blow, D.M. (1974) Biochemistry 13, 4212-4228 Bieth, J. and Frechin, J.C. (1974) Biochim. Biophys. Acta 364, 97--102 Koide, T. and Ikenaka, T. (1973) Eur. J. Biochem. 82, 401---407 Greenfield, N. and Fasman, G.D. (1969) Biochemistry 8, 4108--4116 Koide, T. and Ikenaka, T. (1973) Eur. J. Biochem. 32, 427--431 Ozawa, K. and Laskowski, Jr., M. (1966) J. Biol. Chem. 241, 3955--3961 Gross, E. (1967) Methods in Enzymology 11,238--255, and references therein Hits, C.W.H. (1967) Methods in Enzymology 11,199--203 Light, A. (1967) Methods in Enzymology 1 1 , 4 1 7 - - 4 2 0 Koide, T., Ikenaka, K., Ikeda, K. and Hamaguehi, K. (1974) J. Biochem. (Tokyo) 75, 805--823 Baba, M., Hamaguehi, K. and Ikenaka, T. (1969) J. Biochem. (Tokyo) 65, 113--121 Ikeda, K., Hamaguchi, K., Yamamoto, M. and Ikenaka, T. (1968) J. Biochem. (Tokyo) 63, 521--531 J/rgensons, B. (1973) Biochlm. Biophys. Acta 328, 314--322 Jirgensons, B. (1972) Makromol. Chem. 158, 1--8 Jirgensons, B. and Capetillo, S. (1970) Biochim. Biophys. Acta 214, 1--5 Jirgensons, B., Kawabata0 M. and Capet/llo, S. (1969) Makromol. Chem. 125, 126--135 Jirgensons, B. (1967) J. Biol. Chem. 242, 912--918 Gorbunoff, M.J. (1970) Biochim. Biophys. Acta 2 2 1 , 3 1 4 ~ 3 2 5 Edelhoch, H. and Steiner, R.F. (1963) J. Biol. Chem. 238,931--938 Beychok, S. (1967) in Poly-a-Amino Acids, (Fasman, G.D., ed.), Vol. 1, pp. 293--337, Dekker, New York Barela, T.D. and Darnall, D.W. (1974) Biochemistry 13, 1694--1700 White, F.H., Jr. (1976) Biochemistry 15, 2906--2912
Biochimica et Biophysica Acta, 532 (1978) 327--336 © Elsevier/North-HollandBiomedicalPress
BBA 37832 A CIRCULAR DICHROISM STUDY OF THE CYANOGEN BROMIDE FRAGMENTS OF SOYBEAN TRYPSIN INHIBITOR (KUNITZ)
CLAUDIO TONIOLO, GIAN MARIABONORA, CLAUDIO VITA and ANGELO FONTANA Institute of Organic Chemistry, Biopolymer Research Centre, C.N.R., University of Padova, 35100 Padova (Italy)
(Received June 3rd, 1977) (Revised manuscript received September 29th, 1977)
Summary Fragmentation of soybean trypsin inhibitor (Kunitz) at the level of Met-84 and Met-114 with cyanogen bromide and subsequent reduction of disulfide bridges, S-carboxymethylation and gel chromatography have been employed to obtain in pure form fragments 1--84, 85--114, 115--181, as well as fragment 1--114 with the internal peptide bond 84--85 cleaved. Conformational analysis using circular dichroism in the far-ultraviolet region has been carried out in aqueous solution on the isolated fragments, the native inhibitor, and its reduced S-carboxymethylated derivative. Whereas the native inhibitor does not appear to possess any regular structure, reduction of disulfide bridges and S-carboxymethylation leads to the appearance of some significant regular conformation. Fragments 1--84 and 1--114 are the most prone to assume a regular structure (essentially a ~-conformation), whereas fragments 85--114 and 115--181 are structureless. By using structure-promoting agents (trifluoroethanol and sodium dodecyl sulfate} it has been possible to induce significant levels of a-helix in all the compounds examined. The results obtained appear to support the hypothesis that partial cleavage of the polypeptide chain and reduction of disulfide bonds permit the formation of a more regular structure than exists in the native inhibitor.
Protein conformation is determined by a number of forces, which are often divided into short-, medium- and long-range interactions [1--3]. A well-established experimental approach for elucidating the nature and magnitude of such Abbrevlations: ABC, cyanogen bzomide fragment 1--114; AB-CM, S-carboxymethylated fragment 1--84; C-CM, S-carboxymethylated fragment 85--114; D, fragment 115--181; D-CM, S-carboxymethylated fragment 115--181; SDS, s o d i u m dodecyl sulfate; Hse, homoserine; Tris, tris(hydroxymethyl)aminomethane.
328
interactions is represented by conformational studies of peptide fragments of fully characterized proteins [4--14]. Generally, it has been shown that in aqueous solution peptide fragments contain less secondary structure than the corresponding sequences in the parent protein. The conclusion reached from studies of fragments of myoglobin [4--6], ribonuclease [7--9], nuclease [10-12], hemoglobin [13], and cytochrome C [14] is that in general long-range interactions increase the amount of regular conformation in proteins. In the present paper we report a far-ultraviolet circular dichroism study of the secondary structure of reduced and S-carboxymethylated cyanogen bromide fragments of soybean trypsin inhibitor (Kunitz). This protein was shown by X-ray crystallography to possess little regular structure, most of the polypeptide chain being involved in only approximate pleated-sheet ~-form and with no a-helix [15]. The results reported here indicate that partial peptide bond cleavage and reduction of disulfide bonds of the protein induce the tendency of part of the polypeptide chain to form more regular structure (mostly /3-structure) than exists in the native protein. Materials and Methods Soybean trypsin inhibitor (Kunitz), type I-S, was obtained from Sigma (St. Louis, Miss., U.S.A.). Cyanogen bromide, 2-mercaptoethanol, 2,2,2-trifluoroethanol, dithiothreitol, iodoacetic acid, Tris, and 98--100% formic acid were Fluka (Basel, Switzerland) products and were used without further purification. Urea (Erba, Milan, Italy) was recrystallized from 95% ethanol and only fresh solutions of the reagent were used. Sodium dodecyl sulfate (Fluka) was recrystallized from methanol/benzene. Diethylaminoethyl-cellulose (DE-52) was obtained from Whatman (Maidstone, U.K.) and Sephadex G-50 SF from Pharmacia (Uppsala, Sweden). All other reagents were analytical grade and used without further purification.
Purification of soybean trypsin inhibitor The commercial sample of the inhibitor (200 mg) was purified on a column (2 X 25 cm) of diethylaminoethyl-ceUulose (DE-52) equilibrated with 0.01 M ammonium acetate buffer, pH 6.5 [16]. The column was eluted with a linear gradient of the same buffer from 0.01 to 0.5 M, at a flow rate of 25 ml/h. Fractions of 6 ml were collected and the protein located in the effluent by absorption measurements at 280 nm. The fractions corresponding to the main peak of the protein were combined and then the protein recovered by lyophilization. The protein sample was rechromatographed on the same column [16], eluted in the same conditions, and again recovered by lyophilization. This sample was used in the present study for fragmentation with cyanogen bromide.
Cleavage with cyanogen bromide [17] Purified soybean trypsin inhibitor (100 mg) was reacted with cyanogen bromide (110 rag; approx. 50 equiv.) in 10 ml of 70% formic acid for 24 h in the dark. The reaction mixture was then diluted with water and evaporated in vacuo. The concentrated solution was then applied over a column (3.3 X 140 cm) of Sephadex G-50 Superfine equilibrated and eluted with acetic acid/ formic acid/water (40 : 10 : 50, v/v). The effluent was monitored by absorp-
329 tion measurements at 280 nm. Three peaks of peptide material were obtained, which were shown, by amino acid analysis, to correspond to uncleaved soybean trypsin inhibitor (~ 10%) and fragments ABC and D. The effluents corresponding to the two fragments were concentrated and rechromatographed on the same column. Purity of the peptides was tested by amino acid analysis. Reduction and S-carboxymethylation 1. Soybean trypsin inhibitor. To a solution of 20 mg of purified soybean trypsin inhibitor in 2 ml of 8 M urea in 0.1 M Tris pH 8.2, 3.1 mg of dithiothreitol were added and the pH adjusted to 9.5 with 0.1 M NaOH. The reaction mixture was left at room temperature in the dark for 4 h and then the pH was adjusted to 8.0 with 0.1 N HC1. Iodoacetic acid (15 mg) was dissolved in 0.5 ml of water and the pH adjusted to 8.0 with 0.1 M NaOH and then added to the solution of the reduced soybean trypsin inhibitor. The excess of alkylating agent was removed by the addition of 70 pl of mercaptoethanol and the pH was kept at 7.0 for 15 min. The reaction mixture was directly applied to a Sephadex G-25 Superfine column (2 X 50 cm) equilibrated with 10% acetic acid. The effluent was analyzed at 280 nm.The protein peak was collected and the reduced and carboxymethylated soybean trypsin inhibitor was recovered by lyophilization. 2. Fragment ABC. Reduction and carboxymethylation of fragment ABC were carried out as above. The reaction mixture was then applied to a Sephadex G-50 Superfine column (3.3 X 140 cm) equilibrated with acetic acid/formic acid/water (40 : 10 : 50, v/v). Fragments AB-CM and C-CM were completely separated from each other. The peaks of the fragments were preceded by trace amounts of uncleared fragment ABC. 3. Fragment D. Reduction and carboxymethylation of fragment D were carried out as above. Fragment D-CM was separated by gel filtration on a Sephadex G-25 Superfine column (2 X 50 cm) equilibrated with 10% acetic acid and recovered by lyophilization. Methods
Measurements of absorbance at single wavelengths were carried out with a Hitachi-Perkin-Elmer spectrophotometer, model 139. Continuous spectra were recorded with a Cary 15 spectrophotometer. Circular dichroism spectra were measured with a Cary dichrograph, model 61. Cylindrical fused quartz cells with 0.5- and 1-mm path length were used. The instrument was flushed with prepurified nitrogen before and during the experiments. In the spectra the mean residue ellipticity values [0 ] are reported. They are defined as follows: [0] = (O/lO)(M/lc), where 0 is the measured ellipticity in degrees, l is the path length of the solution in cm, c is the concentration in g/cm 3, and M is the mean residue molecular weight. The latter was taken as 111.1 for soybean trypsin inhibitor, 108.0 for fragment ABC, 112.2 for reduced and carboxymethylated soybean trypsin inhibitor, 109.2 for AB-CM, 107.3 for C-CM, 115.8 for D, and 117.4 for D-CM. The various right-handed s-helical contents of the reduced and S-carboxymethylated trypsin inhibitor
330 and peptide fragments were calculated using the relationship proposed by Greenfield and Fasman [18] on the basis of the ellipticity values at 208 nm. The concentrations of solution were determined by quantitative amino acid analysis of solutions of known absorption and then checked routinely by measurements of absorbance at 280 nm. The pH values were measured with a Metrohm pH meter, model E-510, equipped with a combined glass electrode. Results
Preparation and characterization of fragments The amino acid sequence of soybean trypsin inhibitor contains 181 amino acid residues, and in particular two disulfide bridges (Cys39-Cysa6 and Cys 13~Cys 14s) and two methionine residues (in positions 84 and 114) [19]. The peptide bond between Args3 and Ile 64 is split on interaction with trypsin, giving a modified soybean trypsin inhibitor with a cleaved peptide bond [17,20]. Cyanogen bromide cleavage [21] of the protein yields three fragments, namely 1--84, 85--114 and 115--181. Since fragments 1--84 and 85--114 are linked together via a disulfide bridge, gel filtration on a Sephadex G-50 SF column allows separation of sequences 1--114 (ABC) and 115--181 (D). Reduction and S-carboxymethylation of fragment ABC according to general procedure (ref. 22 and references therein) and passage of the reaction mixture on a Sephadex G-50 SF column allows complete separation of the S-carboxymethylated sequences 1--84 (AB-CM) and 85--114 (C-CM). Native soybean trypsin inhibitor and fragment D were also reduced and S-carboxymethylated, and the derivatives (reduced and carboxymethylated soybean trypsin inhibitor and D-CM, respectively) were purified by gel filtration. The general scheme of fragmentation of native soybean trypsin inhibitor is shown in Fig. 1. The nomenclature of the fragments used is similar to that proposed by Koide and Ikenaka [17]. The purity of soybean trypsin inhibitor, its fragments and their S-carboxymethylated derivatives was tested by amino acid analysis (Table I). Analyses were in good agreement with theory [19]. The low values of leucine residue found only in the intact inhibitor, fragment D, and their reduced and S-carboxymethylated derivatives confirm that some molecules of the inhibitor lack C-terminal leucine [17]. The low recovery on the analyzer of valine and isoleucine residues should be related particularly to the presence of Val-Val, ValIle and Ile-Ile sequences, which are known to be difficult to hydrolyze [17,19, 23]. The above hypothesis is in part confirmed by the observation that the values of isoleucine residue are low only in the peptides where Val-Ile and IleUe bonds occur (soybean trypsin inhibitor, its reduced and carboxymethylated derivative, and fragments ABC and AB-CM}.
Circular dichroism studies Effect of pH. The far-ultraviolet circular dichroism spectra of native soybean trypsin inhibitor and fragments ABC and D at pH 1.0, 7.0 and 11.0 are shown in Fig. 2. The curve recorded for native soybean trypsin inhibitor at pH 7.0, exhibiting a positive maximum at 226 nm and a negative maximum near 200 nm, is in agreement with those reported in the literature where they have been
331 1 Asp
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I
84
85
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181
114 115 Met - Asp
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84 H se
SCH2COOH114 85 I N se Leu C -CM
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181 Leu
D-CM
Fig, 1. The schematic representation of the f r a g m e n t a t i o n of s oybe a n t ryps i n inhibitor.
interpreted as indicative of an aperiodic conformation [24--32]. The conformation of native soybean trypsin inhibitor does not appear to be significantly affected by the pH of the solution (Fig. 2A) [25,26,30]. Fragment ABC in 20 mM phosphate buffer, pH 7.0, shows a circular dichro-
TABLE I AMINO ACID COMPOSITION OF SOYBEAN TRYPSIN INHIBITOR, ITS REDUCED AND CARBOXYM E T H Y L A T E D D E R I V A T I V E AND CYANOGEN BROMIDE FRAGMENTS ABC, AB-CM, C-CM, D AND D-CM Values are not corrected for losses during acid hydrolysis. Theoretical values s.re shown in parentheseL Amino acid
STI *
STI-CM *
ABC
AB-CM
C-CM
D
D-CM
Aspartic acid Threonine Serine Glutamic acid Prollne Glycine Alanine Half-cystine
26.8 (26) 7.1 (7) 10.8(11) 18,3 (18) 10.6(10) 15.6 (16) 8,5 (8) 4.3 ( 4 ) -12.4(14) 2.1 (2) 12.6 (14) 14.3(15) 3.9 (4) 8,6 (9) 10.1 (10) 2.3 (2) 8.8 (9)
26.2 (26) 6.6 (7) 10.2(11) 18.6 (18) 10.7(10) 16.4 (16) 7.7 (8)
13.0 (13) 6.0 (6) 7.2 (7) 9.9 (10) 8.5 (8) 12.0 (12) 7.0 (7)
10.5 (10) 4,8 (5) 5,9 (6) 6,6 (6) 5,4 ( 5 ) 9.2 (9) 4.9 (5)
3.1 (3) 1.0(1) 1.0(1) 4.2 (4)
13.2 (13) 1.3 (1) 3.6 (4) 8.1 (8)
13.1 (13) 0.9 (1) 3.7 (4) 8.3 (8)
CM-cysteine
Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine LyJ~ne Histidine Arginine
--
3.8 (4) 10.8(14) 1.9 (2) 12.7 (14) 14.3(15) 3.9 (4) 8.9 (9) 10.2(10) 2.0 (2) 9.3 (9)
2.0
(2)
-6.8 (8) . . 9.6 (11) 8.7 (9) 2.6 (3) 5.0 (5) 3.9 (4) 1.1 (1) 5.3 (5)
3.6(3)
2.3
(2)
2.1
(2)
3.0 (3) 2.0(2)
4.0 1.2
(4) (1)
4.3 1.2
(4) (1)
Ne_~]i~Ible
2.4
(2)
--
-5.9
(6)
1.8 4.5
(2) (6)
(9) (7) (3) (5)
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3.2 5.3 1.0 3.9
(3) (6) (1) (4)
2.9 5.2 1.1 3.6
(3) (6) (1) (4)
(2) (1)
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5.7 1.2
(6) (1)
6.4 1.0
(6) (1)
(5)
--
3.8
(4)
4.1
(4)
--
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7.8 6.9 2.7 4.7 2.2 1.0 4.8
.
* STI, s o y b e a n t r y p s i n i n h i b i t o r ; STI-CM, S - c a r b o x y m e t h y l a t e d s o y b e a n t r y p s i n inhibitor.
332
i
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Fig. 2. F a r - u l t r a v i o l e t c i r c u l a r d i c h r o i ~ n s p e c t r a o f s o y b e a n t r y p s i n i n h i b i t o r and its c y a n o g e n b r o m i d e fragments A B C and D at r o o m t e m p e r a t u r e at p H 1 . 0 . . . . , in 2 0 m M s o d i u m p h o s p h a t e buffer• p H 7.0 • and at pH 11.0 ...... . A, native protein; B, f r a g m e n t A B C ; C, f r a g m e n t D.
ism spectrum with a negative band centered at approx. 205 nm (the corresponding mean residue ellipticity values is --5500 _+300 degree • cm 2 . dmol-') and a prominent shoulder near 217 nm (Fig. 2B) [24]. The characteristics of the curve indicate the presence of some significant regular structure. The conformation of fragment ABC is affected by acid and alkali, since a strong negative Cotton effect is generated and centered at 210--215 nm at pH 1.0 and 11.0. Conversely, fragment D appears to possess little regular structure in neutral, acid or alkaline aqueous solution (Fig. 2C), since regularly increasing negative ellipticity is shown in the region 250--200 nm with maxima near 200 nm [24]. Figure 3 shows the CD curves recorded at pH 1.0, 7.0, and 11.0 of reduced and c a r b o x y m e t h y l a t e d soybean trypsin inhibitor, AB-CM, C-CM and D-CM. The circular dichroism spectrum of reduced and carboxymethylated soybean trypsin inhibitor (Fig. 3A) at pH 7.0 is characterized by a negative band centered near 206 nm with an ellipticity value of --6500 +-300 degree • cm 2 • dmo1-1 and a shoulder near 215 nm [30]. Fragment AB-CM in aqueous buffer, pH 7.0, shows a negative band centered at 214 nm with an ellipticity value of --4500 + 300 degree • cm 2 • dmol -~ (Fig. 3B). The presence of regular structure in both cases is evident and the characteristics of the circular dichroism pattern of fragment AB-CM could be related to the occurrence of a significant extent of ~-conformation. Changing the pH of the solution from neutral to acid or alkaline region appears to induce conformational transitions both in reduced and c a r b o x y m e t h y l a t e d inhibitor and AB-CM. At acid pH the content of ~ o n f o r m a t i o n in reduced and c a r b o x y m e t h y l a t e d inhibitor is increased, whereas alkaline pH is effective in destroying the periodic structures. Fragments C-CM and D-CM exhibit CD spectra in neutral as well in acidic or alkaline solution indicative of peptide chains in an essentially aperiodic form. The state of aggregation of the various fragments under the experimental conditions employed for the circular dichroism measurements may have an
333 0 I { -2
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-8
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i
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Fig. 3. F a r - u l t r a v i o l e t c i r c u l a r d i c h r o i s m s p e c t r a of r e d u c e d a n d S - c a r b o x y m e t h y l a t e d i n h i b i t o r a n d fragm e n t s AB-CM0 C-CM a n d D-CM a t r o o m t e m p e r a t u r e at p H 1.0 ( . . . . ), in 20 m M s o d i u m phosphate b u f f e r , p H 7.0 ( ), a n d a t p H 1 1 . 0 ( . . . . . . ). A, r e d u c e d a n d c a r b o x y m e t h y l a t e d i n h i b i t o r ; B, fragm e n t AB-CM; C, f r a g m e n t C-CM; D, f r a g m e n t D-CM.
influence on the spectral properties observed. Altough no molecular weight estimations were carried out to verify conclusively this point, it has been shown that circular dichroism data are independent of peptide concentration within the limits of experimental error. In particular, concentration effects have been studied in more detail with fragments ABC and AB-CM, which appear to possess some structure in aqueous solution (Figs. 2 and 3). Presumably, at the low concentrations used in the circular dichroism experiments (approx. 0.1 mg/ ml) the peptides are mostly in the monomeric form.
Effect of trifluoroethanol and SDS Native soybean trypsin inhibitor, its reduced and S-carboxymethylated derivative and all the fragments herewith investigated give in 98% trifluoroethanol a family of circular dichroism curves resembling that of helical polypeptides, i.e., with two negative maxima near 222 and 208 nm [34]. In particular, in each case the band at 208 nm is about 10% deeper than that at 222 nm. The amount of a-helix generated by the haloalcohol was calculated from the relationship proposed by Greenfield and Fasman [18] which makes use of the ellipticity value at 208 nm. The resulting percentages are reported in Table II. Alkyl sulfates with a long hydrocarbon chain, like SDS, are also effective in inducing the formation of a-helix in soybean trypsin inhibitor [27--31]. The effect of SDS was examined at 2% concentration and at pH 2.2, where the interaction between the anionic detergent and the polypeptide chain is maxi-
334 T A B L E II P E R C E N T O F R I G H T - H A N D E D { x - H E L I C A L C O N F O R M A T I O N IN S O Y B E A N T R Y P S I N I N H I B I T O R , ITS REDUCED AND S-CARBOXYMETHYLATED DERIVATIVE AND CYANOGEN BROMIDE F R A G M E N T S A B C , A B - C M , B-CM, D A N D D-CM C a l c u l a t i o n s w e r e m a d e a c c o r d i n g t o G r e e n f i e l d a n d F a s m a n [ 1 8 ] u s i n g t h e e l l i p t i c i t y v a l u e a t 2 0 8 rim. Compound
98% trifluoroethanol *
2% S D S pH 2.2
Soybean t~ypsin inhibitor CM-derivative of the inhibitor ABC AB-CM C-CM D D-CM
57 57 47 45 14 52 28
25 21 21 20 10 17 11
* 9 8 % t r i f l u r o r o e t h a n o l in 2 0 m M s o d i u m p h o s p h a t e b u f f e r , p H 7.0.
mal [28]. Comparatively to trifluoroethanol lower percentages of a-helix are observed in all compounds examined (Table II). Discussion
Quantitation of secondary structure of proteins can be obtained from an analysis of their CD spectra in the far-ultraviolet region [18]. However, there are several limitations in all the proposed methods of calculation [35] and the estimates should often be regarded as only qualitative. Nevertheless, farultraviolet circular dichroism measurements are useful in detecting structural properties and conformationai transitions of polypeptide chains. The far-ultraviolet circular dichroism spectrum of soybean trypsin inhibitor is indicative of an aperiodic conformation (Fig. 2A) in agreement with its crystal structure determined by X-ray diffraction [15]. The protein appears in fact as a sphere of approx. 35 A in diameter, made by criss-crossing loops wrapped around a core of hydrophobic side chains, with no a-helix and only irregular ~-pleated-sheet structure. The basic observation made in the present work is that fragmentation of the peptide chain of soybean trypsin inhibitor as well as reduction of disulfide bonds favor the formation of a significant amount of regular secondary structure in part of the inhibitor chain which is absent in the native molecule. In fact, the far-ultraviolet circular dichroism patterns of ABC, reduced and carboxymethylated inhibitor, and AB-CM suggest the existence in aqueous solution of polypeptide chains containing structurally periodic segments. We have found that the pH of the solution induces conformational transitions in some of the examined species, at variance with native inhibitor whose conformation is practically unaffected on going from neutral to acid or alkaline media [25, 26,30]. Since the N-terminal fragment (ABC) of the inhibitor is prone to assume a regular structure, while the C-terminal fragments C-CM, D and D-CM are essentially structureless, we propose that also in the reduced and S-carboxymethylated protein the structured region of the chain would be the N-terminal
335
one. This finding could bear upon the possibility role of the amino end of the chain in initiating protein folding [3]. The haloalcohol trifluoroethanol and the detergent SDS induce the onset of a-helical conformation into a considerable extent in all the compounds examined, as indicated by the circular dichroism spectra with negative maxima near 222 and 208 nm. Although in a few cases some deviation was observed from the standard a-helix spectrum given, for example, by Greenfield and Fasman [18], the mean residue ellipticity values at 208 nm were used to calculate the percentages of a-helix attained in the presence of trifluoroethanol and SDS. The extent of a-helix of reduced and carboxymethylated inhibitor in trifluoroethanol is substantially higher than in the three reduced and S-carboxymethylated fragments taken together. This could be related to end-group effects produced by cleavage of the 84--85 and 114--115 peptide bonds by cyanogen bromide, and/or possibly to the occurrence of long-range interactions [3] stabilizing helical segments in reduced and carboxymethylated inhibitor. However, the interpretation of all the figures reported in Table II is not straightforward and it does not seem to us worth a detailed discussion. This study may contribute to the comprehension of the mechanism of folding of proteins. In considering the structure of a protein in aqueous solution, short-, medium- and long-range interactions along the peptide chain are considered as determining the specific folded structure of the molecule [3]. Usually, short- and medium-range interactions are thought as being the most important and these are employed to predict secondary structure of proteins. Sometimes unsuccessful or poor predictions of protein structures result from the application of oversimplified rules which do not take into account longrange interactions. Analogously, the extent of periodic structure in fragments of globular proteins has usually been found to be far below the expected values and also this change in conformation has been related to the occurrence of long-range interactions which increase the amount of regular structure of the native polypeptide chain. A possible, although not the only, explanation of the data on fragments of a protein with complete aperiodic structure discussed here is that long-range interactions, though in most cases enhancing the percentage of periodic conformation of a peptide molecule, in certain limited cases could prevent the chain from assuming its maximal degree of regular structure. The present results parallel those recently reported by White [26] who was able to demonstrate that reduction of disulfide bonds in lysozyme and subsequent S-alkylation favors the attainment of more ~-structure than exists in the native protein. Acknowledgments The authors wish-to thank Mr. M. Zambonin, Mr. S. Fioretto Da Rin and Mr. F. Miozzo for expert technical assistance. References 1 Tanaka, S. and Scheraga, H.A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3 8 0 2 - - 3 8 0 6 2 Wetlaufer, D.B. an d Ristow, S. (19"/3) Annu. Rev. Biochem. 42, 135--158
336 3 4 5 6 7 8 9 I0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
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