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Conformational studies of the α-helical proteins from wool keratin by c.d.
Conformational studies of the a-helical proteins from wool keratin by c.d. Takayuki Amiya, Kanji Kajiwara, Takeaki Miyamoto* and Hiroshi lnagaki Institute l}~r Chemical Research, K yoto Unit~ersity, Uji, K yoto 611, Japan
(Received 14 September 1981; revised 23 October 1981) The co,/brmation and col?/brmational chcmqe ~?[ wool keratin S-carboxymethylated low-sulphur proteins (SCMKA), which are ~-helieul./ibrous proteins, hal'e been int'estigated in aqueous .solution by means ~71e.d. Comparison,s ~?Jl~arious methods proposed fi)r c.d. analysis ofprotein secondary structure are made using leastsquares curt'ez/ittin,q ~?]the obsereed c.d. spectra ~?[SCMKA with a linear combination ~?[the correspomlin.q r¢~fbrence spectra ~?Jsecondary structures. It has beenJbund that (i) the most sati,~/Uctory results ure obtained with the method ' 3 which takes into account the [~-turn contribution; (ii) S C M K A is 52 54'!o cuhelical in water and has little [4:[~rm:(iii) the addition g/n-propanol produces, eeen at higher concentrations ~?[n-propanol, little change in spectra with respect to helical character in water; (iv) S C M K A undergoes a thermally-induced eo~Ibrmational tralTsition Ji'om ~-helix to random c'oil around 50 C: and (l:) S-aminoethylated low-sulphur proteins with positieely charged proteetinq ,qroups are ~ 50°0 cuhelieal in water, which is similar to S C M K A, showing that the protecting groups introduced in the low-sulphur proteins have little e[J~ct upon their confi)rmation in water. Keywords: Optical chemical analysis; keratin; fibrous protein; secondary structure; low sulphur proteins: Scarboxymethylkerateine
Introduction ~-Keratins such as wool or animal hair consist of ordered filamentous units, termed 'microfibrils' embedded in a less ordered nonfilamentous matrix l"2. Keratin proteins can be obtained as soluble derivatives after rupture of the disulphide bonds, and can be divided into three main fractions: low-sulphur, high-sulphur and high-glycine fractions 1-3. Proteins of the last two fractions do not assume a helical structure in aqueous solution and are considered to originate chiefly from the matrix. The proteins in the low-sulphur fraction, however, are partly helical in aqueous solution over a wide range of pH values, and are considered to originate from the microfibrils. Proteins from ~-keratins are studied in soluble form by reduction of the disulphide bonds and protection of the resulting thiol groups with iodoacetic acid to form Scarboxymethylkerateines (SCMK)t. Considerable progress has been made on the primary structure by chemical and physicochemical characterization of wool S C M K proteins 3. Until now, however, studies on the conformation and conformational transition of S C M K proteins in solution have relied mainly on the MoffittYang parameter bo given by o.r.d, measurement 4-6, except for our recent c.d. studies on matrix proteins 7'8. C.d. is a powerful technique for studying protein conformation in solution. When a set of reference spectra for secondary structure, a-helix, /~-form, /~-turn and unordered form are given, the content of each secondary structure in a protein can be estimated by using a leastsquares curve-fitting of the observed c.d. spectra to a * To whom correspondence should be addressed. t The term 'kerateine' is generallyused for the reduced form of keratin. 0141 8130/82/030165q38503.00 Q1982 Butterworth & Co. (Publishers) Ltd
linear combination of the corresponding reference spectra 9-14. The present paper deals with conformation and conformational change of S-carboxymethylated lowsulphur proteins (SCMKA), which are co-helical proteins from wool, in aqueous solution by c.d. The problem was divided into three objectives. The first was to find the most satisfactory set of reference spectra for the c.d. analysis of S C M K A secondary structure. Comparisons were made of three representative methods proposed for c.d. analysis of protein secondary structure 9 ' ' t t 3 The second was to investigate the conformational changes is S C M K A during alcohol and thermal denaturation. Analysis of the observed c.d. spectra was performed with the most satisfactory set of reference spectra. In the case of SCMK, a large amount of negatively charged groups is introduced into the cysteinyl residues because of the high cystine residue content of wool keratin. The third objective was to examine the influence of these charged groups on the conformation of S C M K A in aqueous solution. The conformation of S C M K A was compared with that of S-aminoethylated low-sulphur proteins (SAEKA) whose protecting groups are positively charged.
Experimental Materials
Merino 64 wool was purified according to the standard method 15. Three-times recrystallized c~-chymotrypsin from bovine pancreas was purchased from Sigma Chemical Co., USA. Deionized water was used throughout. All other chemicals were of reagent-grade and were used without further purification.
Int. J. Biol. Macromol., 1982, Vol 4, April
165
~-ftelical proteins li'om wool keratin: Tukayuki Amiya el al. Table 1 Amino acid composition ofSCMKA, SCMK-HF and SAEKA from Merino wool" Amino acid Lys His Arg AECys j' CMCys' Asp Thr Scr Glu Pro Gly Ala Cys/2 Val Met lieu Leu Tyr Phe
SCMKA SCMK-HF 3.0 0.5 6.9
5.7 0.7 7.1
7.2 8.3 5.2 9.1 16.1 4.7 8.4 6.5 0.0 4.9 0.4 3.6 8.8 3.9 2.5
3.1 11.8 3.4 6.2 20.9 1.8 2.8 8.1 6.3 0.3 4.4 14.7 0.8 1.8
SAEKA
Native
3.0 (t.5 6.9 6.9
2.9 0.~ 6,9
8.1 4.8 9.5 15.3 4.6 8.6 6.0 0.0 5.7 0.4 3.1 9.4 4.2 2.8
5.9 6.2 10.4 11.8 10.1 5.6 5.1 11.1 6.1 0.5 3.2 7.3 3.7 2.6
Values given as mol per 100 mol amino acids ~' 2-Ammoethylcysteine Carboxymetlaylcysteine
Dowling TM. The purification o f S C M K HF was repeated twice by reprecipitation at pH 4.0 from salt solution. The precipitate was dissolved in aqueous sodium borate solution. After dialysis against deionized water, the solution was freeze-dried. The molecular weight of the SCMK-H F was estimatcd by the use of a Sephadex G-75 colunm in a buffer containing 8 M urea at pH 7.4 as described previously ~' The sample consisted of three subfractions, where the major peak followed the peaks of two minor fractions of molecular weight > 2 x 104. Such an elution pattern was very similar to that for the helical fragments reported by Crewther and Dowling TM. The similarity m amino acid composition between our sample and that of Crewther et a/. was also confirmed as described later. In the present study, the S C M K - H F sample was used for the c.d. measurements without further fractionation, i.e. without removal of small amounts of higher molecular weight subfractions.
Esterflication o1 S C M K A Esterification of SCMKA was carried out by treating SCMKA with methanolic-HC1 for 24 h at 20 C ~'. The csterified SCMKA redissolved in 8 M urea solution was dialysed against deionized water and then freeze-dried.
C.d. spectra Preparation of low-sulphur protein derivatives The low-sulphur proteins of S-carboxymethylkerateine {SCM KA) were prepared from reduced wool according to the procedure of Dowling and Crewther ~s. This involved the extraction of wool with 2-mercaptoethanol at pH 10.5 in the presence of 8 M urea, the alkylation of extracted proteins with iodoacetic acid, and the precipitation of the low-sulphur proteins from 0.5 M KC1 solution at pH 4.4. The reprecipitation from salt solution was repeated three times to remove high-sulphur and high-glycine proteins. Finally, the sample fraction redissolved in sodium borate was dialysed against deonized water and then freeze-dried. The average molecular weight of SC M KA is known to be about 5 x 104 as determined by gel filtration ~' The S-aminoethylated derivative from reduced wool (SAEK) was prepared by a similar procedure to that of SCMK except that 2-bromoethylamine was used as an alkylating agent instead of iodoacetic acid ~7. The alkylation was continued by addition of 2bromoethylamine until the nitroprusside test for thiol became negative. The low-sulphur protein fraction (SAEKA) was obtained by fractional precipitation of SAEK by the procedure of Ito~ 7. The sample SAEKA was further purified by repeating precipitation from the salt solution several times to remove traces of other protein fractions. These fractionation and purification procedures were confirmed to be satisfactory from amino acid analysis and gel filtration chromatography of SAEKA on a column of Sephadex G-200 in 8 M urea ~7. Amino acid analysis also ensured that the cystinyl residue had been converted quantitatively to a 2-aminoethylcysteinyl residue and that the other residues remained unmodified Isee Table 1).
Preparation olhelix-richfi'agmentsJ?om S C M K A Helix-rich fragments (SCMK HF) from S C M K A were prepared by limited digestion of S C M K A with ~,chymotrypsin according to the method of Crewther and
166
Int. J. Biol. Macromol., 1982, Vol 4, April
C.d. spectra were taken on a Jasco J-20 spectropolarimeter in a thermostatically controlled 1 mm .jacketed cell. The c.d. data were expressed in terms of the mean residue ellipticity, [01, in deg cm 2 drool ~. The mean residue weight of each derivative was calculated from its amino acid composition. For c.d. measurements of the films, the sample solution with a different solvent was layered directly on a quartz disc used for the measurement to prepare the film. C.d. spectra were expressed in terms of the difference in absorbance for left and right circularly polarized light. The protein concentration was determined by weight since we had a sufficient quantity to prepare a stock solution of known concentration.
Amino acid analysis The sample proteins were hydrolysed in racuo at 110 C for 24 h with 6 N HCI and 2 mM phenol. The amino acid content of the hydrolysates was estimated using a Hitachi KLA-5 amino acid amdyser. The results were expressed in terms of tool per 100 tool amino acid. Table 1 summarizes the amino acid compositions of SCMKA, SCMK-HF, SAEKA and native Merino wool.
C.d. analysis In general, the c.d. spectrum of a protein is expressed m terms of a linear combination of the respective secondary structures contained in the protein. Thus the mean residue ellipticity, [0]~, at an arbitrary wavelength, 2, is given by:
[o]~. =
~ } F o ] ~..j
t l)
i where [0] ,..j denotes the reference values for the secondary structure of j, and./i its corresponding content. When a set of reference spectra for secondary structures are given, the content Ji of each secondary structure in a protein is
~-Helical proteins from wool keratin: Takayuki Amiya et ul. estimated according to equation (1) so as to yield the bestfit linear combination of reference spectra with respect to the experimental c.d. spectrum. Several methods have been proposed to analyse the spectrum 9- ~4, where the difference is due to the choice of reference spectra for each secondary structure. In the present study, c.d. analysis of S C M K A secondary structure was performed by following three representative methods. The first method (method I), which may be called the Greenfield Fasman method 9, uses the c.d. spectra of synthetic polypeptides in s-helix, //-form and random form, as reference spectra. The second (method II) is that ofChen et al. ~~ in which the calculated c.d. spectra of each secondary structure of certain proteins whose structures were estimated by X-ray data, are used as reference spectra of three structural elements. In these two methods, it is assumed that proteins contain only two types of regular structural elements: c~-helix and//-form. The third method tested (method III), is that of Yang and coworker in which the//-turn contribution is taken into account 13. Here the reference spectra (data with a 1 nm interval) of the a-helix,/#form, fl-turn and unordered form, were kindly provided by Prof. J. T. Yang. For j) determination, the following two constraints were introduced: (1) the sum of the contents of all structural elements is unity, i.e. YJi = 1, and (2) the content of each structural element is positive, i.e.Ji/> 0. The fitting was performed on a computer F a c o m M160AD by a least-squares method with 1 nm intervals, in the region 190 to 240 rim. The goodness of fit between experimental and calculated values was evaluated in terms of the root mean square error (STD) as: STD = [Z(A0~.)2/(N - i)] 1,2
c.d. spectrum characteristic of a-helix, having a positive band at 192 nm and two negative bands at 209 and 222 nm. It is characteristic of a-keratin that the low-sulphur protein fraction is largely a-helical and that its helical content is relatively high compared with that of globular proteins. The S C M K - H F sample shows a similar c.d. spectrum, but it is clear from the magnitude of the negative ellipticity bands at 209 and 222 nm that S C M K H F contains a higher content of a-helix than SCMKA. Average number of residues per helix The following two factors should be taken into consideration when estimating the secondary structure of a-keratin proteins: (1) c~-keratin consists of a characteristic coiled-coil a-helical structure 2~, and (2) the reference c.d. spectra of a-helix in methods II and III depend on the average number of residues per helix 11"13 X-ray diffraction studies on S C M K A and its helical fragments from wool indicate that these proteins consist mostly of a coiled-coil a-helical structure in the solid state22 24, while analyses of the primary structure for the helical fragments have revealed a repetitive pattern of the type expected from a-helical proteins with a coiled-coil structure a'23. We may predict that S C M K A and S C M K H F might assume a similar coiled-coil a-helical structure in aqueous solution3'% and if so the difference in the c.d. spectrum between a-helix and coiled-coil a-helix should be examined. Recently, Kontani 2s has calculated the theoretical c.d. spectra of these two a-helices, and showed that the c.d. spectrum of coiled-coil a-helix differs little from that of a-helix. This means that the reference spectra of a-helix may be used in the estimation of secondary structure of proteins containing a coiled-coil a-helix.
(2)
where A0;. is the difference between the experimental and calculated mean residue ellipticity at wavelength ), N the number of experimental data, and i the number of reference spectra used for the fitting (i.e. 3 or 4 in the present cases). Since wavelengths were taken with 1 nm intervals from 190 to 240 nm, N = 5 1 . In addition to the 'constrained' method mentioned above, the computation was also per,formed by an 'unconstrained' least-squares method 2°, i.e. without Eli = 1, but with .]j/> 0. The results of analyses using the unconstrained method provide a simple criterion for the validity of a set of reference spectra employed, because the goodness of fit can be evaluated in terms of STD as well as the deviation of Zli from unity according to Baker and lsen berg ~4,2 o.
,
,
,
,
t
t
,
,
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I
,
6
i
-( 0
E
4
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E
u
2 "0 v ~T
0'
0
Results and discussion X
C.d. spectra of S C M K A and S C M K - H F in aqueous .solution S-Carboxymethylated low-sulphur proteins from wool keratin (SCMKA) have an isoelectric point at pH 4.4 and are soluble in aqueous solution when the pH value exceeds 4.4. Fiyure 1 shows the c.d. spectrum of S C M K A in aqueous solution at pH 6.7, ,together with the c.d. spectrum of helical fragments ( S C M K - H F ) prepared by partial proteolysis of S C M K A with a-chymotrypsin. The spectra of these proteins in aqueous solution were almost independent of pH in the range 6- 10. S C M K A shows a
- 2
-~1
I
200
220
!
I
240
I
260
X (rim) Figure 1 C.d. spectra of SCMKA (
) and SCMK-HF
( • - ) in water at pH 6.7
Int. J. Biol. Macromol., 1982, Vol 4, April
167
:~-Helical proteins.t?om wool keratin: Takayuki Amiya et al. Comparison ql" methods Table 4 shows the results of c.d. analysis o b t a i n e d for
The reference value of ~-helix in m e t h o d s I1 and 1II d e p e n d s on the average n u m b e r of residues per helix, n, t h r o u g h the following empirical equation9:
[0];~,= [0J,~(l
k/~0
S C M K A by the three m e t h o d s under the u n c o n s t r a i n e d condition. The calculations were carried out with h = 90. In the case of m e t h o d I, h is effectively infinite. The s u p e r i o r i t y of each m e t h o d has been discussed on the basis of the g o o d n e s s of fit between the e x p e r i m e n t a l and calculated c.d. spectra for S C M K A . The most satisfactory results were o b t a i n e d with m e t h o d I I l where a protein was a s s u m e d to c o n t a i n three types of regular structural elements including fl-turn. In Table 5 are s u m m a r i z e d results of the c.d. analysis of SCMKA under c o n s t r a i n e d conditions. Fiqure d e m o n s t r a t e s e x p e r i m e n t a l as well as calculated c.d. spectra with the respective sets of reference spectra. M e t h o d l l I gives the best fit to the observed c.d. spectrum of S C M K A . T h u s the s e c o n d a r y structure of S C M K A in a q u e o u s solution was estimated to be c o m p o s e d of 53'~,, c~helix, 3'}i, fi-form, 1470 fl-turn and 300o u n o r d e r e d form. The same calculation p r o c e d u r e was applied to S C M K H F and the s e c o n d a r y structure was found to be c o m p o s e d of 79% e-helix, 0'?~,fi-form, 171, fi-turn and 20,,, u n o r d e r e d form. Table 5 also shows that the helix content of S C M K A is almost i n d e p e n d e n t of the n a t u r e of the reference spectra chosen. Previously, H a r r a p 6 estimated the helix content of S C M K A from wool and its helical fragments using the Moffitt Yang p a r a m e t e r b o of synthetic p o l y p e p t i d e s as reference. The values of c~-helix c o n t e n t of S C M K A and S C M K - H F by the present c.d. analysis are very close to
(3)
where the superscript ,~,c,refers to a helix of infinite length and k is a chain-length i n d e p e n d e n t p a r a m e t e r (3.5 > k >2.5). The average n u m b e r h in g l o b u l a r p r o t e i n s has been k n o w n to be ~ 9 1 0 ( R e f s l l a n d l 3 ) . l n t h e c a s e of w o o l keratin, recent studies on the helical fragments from S C M K A have suggested that the p o t e n t i a l l y helical units of S C M K A would have c o n s i d e r a b l y larger h than g l o b u l a r proteins 23'2<27. However, we have no conclusive figure for n of the true helical units of SC M K A in a q u e o u s solution 3. In the present c.d. study, it was necessary to examine n for S C M K A and S C M K - H F from wool. W e have tried to evaluate n for S C M K A and S C M K - H E from the value of [( ] , which gives a best fit to the o b s e r v e d c.d. spectra. F o r this purpose, the s a m p l e S C M K - H F , which originates from the helical region of S C M K A , was used and c o m p a r i s o n s were m a d e with different fi values, with different reference values of ~-helix. It should be m e n t i o n e d here that the n - d e p e n d e n t term c o n t r i b u t e s < 5 , to the total [()]h value when h > 5 0 , as can be seen in e q u a t i o n (3). Tables 2 and 3 show the results of c.d. analysis of S C M K - H F with different h values by m e t h o d s lI a n d lII, respectively. The calculations were carried out u n d e r the u n c o n s t r a i n e d condition. A best calculated s p e c t r u m should be the one where the value of Z/i is nearly equal to unity a n d the value of S T D is the least. The results showed no significant difference in the values of S T D between the n values. However, the other criterion, i.e. ZI}, implied that the value of n should be far larger than 9. The same conclusion was also d r a w n from the calculated spectra of S C M K - H F under the c o n s t r a i n e d condition. T h u s the subsequent c.d. analyses of S C M K and S C M K - H F were carried out with n = 90.
Table 4 C.d. analysis of SCMKA secondary structure with wtrious reference spectra under the unconstrained condition
Table 2 C.d. analysis of S C M K - H F secondary structure with different n values by method II under the unconstrained conditions"
Sample
n
.li,
./)~
.I~:
ZI~
10 "~x STD b
SCMK-HF
9 20 50 90
1.110 0.996 0.905 0.881
0.079 0 0 0
0.466 0.409 0.364 0.353
1.655 1.405 1.269 1.233
0.210 0.206 0.205 0.209
"/, See text for details
Root mean square error (deg c m
Table 3
2
.lu
.li~
.11
1 II IIl
0.523 0.538 0.524
0.181 0 0 0.132
1}~
El i
10 ~ x STD"
0.112 0.330 0.280
0.816 0.868 0.936
0.176 0.103 0.069
" Root mean square error (deg cm2 dmol 1), defined in text Table 5 C.d. analysis of SCMKA secondary structure with various reference spectra under the constrained condition
Method
.fiJ
./)~
1 ll Ill
0.495 0.540 0.529
0.318 0.100 0.030
dmol L), defined in text
.11
.I~
10 4 × STD"
0.139
0.187 0.360 0.302
0.187 0.136 0.070
Root mean square error (deg cm 2 dmol t), defined in text
C.d. analysis of S C M K - H F secondary structure with different h values by method I11 under the unconstrained condition"
Sample SC M K-H F
n
.Iit
.I),
.I,
/~
Z l)
10 ~ x STD"
9
1.131
1)
0.251
0.417
1.799
0.135
20
0.979
0
0.173
0.367
1.519
0.138
50
0.890
0
0.153
0.330
1.373
0.140
90
0.866
0
0.146
0.321
1.333
0.13(/
See text for details ~' Root mean square error (deg cm 2 drool ~), defined in text
168
Method
Int. J. Biol. M a c r o m o l . , 1982, Vol 4, April
2-Helical proteins fi'om wool keratin: Takayuki Amiya et al. a
b-
#"
F~
Method Ill- ~
-6
3
i o
-2 L , 200
220
2aO
200
22-0
2L.O
200
22G
"2c.0
,
3
X(nm)
Figure 2 Comparison of the experimental and computed c.d. spectra of SCMKA in water at pH 6.7. Experimental ( -1; computed ( ): difference between the experimental and computed spectra (.... ). (a) method I; (b) method II; (c) method III those found by Harrap. Such an agreement may be attributed to the fact that the helix part of low-sulphur wool proteins is lengthy. However, it is clear that the reference spectra of method I based on synthetic polypeptides tend to considerably overestimate the Ifform content as much as 32°0, whereas other reference spectra based on globular proteins yield values < 10!?i,. Consequently, the content of unordered form was 19% with method I and ~30'!,, with other methods. In the case of reduced lysozyme 28, more satisfactory results were obtained with the reference spectra of poly(klysine) rather than those based on globular proteins. It is of interest to note that the opposite was true for reduced wool keratin although it is supposed that, unlike globular proteins, the long helix of the low-sulphur proteins from wool has an extended structure so that their shape is more like synthetic polypeptides rather than globular proteins. A great advantage of method Ill is that inclusion of the /~-turn term may improve the estimates of the/Lform and does not affect the estimates of the c~-helix~3. The present results indicate that c.d. analysis by method III is also valid for c~-helical proteins from woo1 and can explain the observed changes in their conformation in terms of the ehelix and /~-form. With regard to the /#turn, however, Chang et al. ~3 pointed out that the calculated contents were not correlated with X-ray results even for the globular proteins. This might be due to the fl-turn reference spectrum chosen. The reference spectrum by Chang et al. was not experimentally measured but extracted from different proteins and is very different from the spectra of/?-turns reported recently by Brahms and Brahms ~4. Thus the /?-turn content estimated for S C M K A would also be uncertain. However, the uncertainty of the calculated /?-turn content does not interfere with the study of the conformation and conformational changes of S C M K A in terms of the ~helix and/~-form.
SC M KA conJormation in alcoho~water mixtures Aqueous alcohol solutions, especially aqueous npropanol, are known to cause marked swelling of wool fibre 29. It is of interest to examine the conformation of low-sulphur proteins in alcohol-water mixtures. This section deals with the effect of n-propanol on the conformation of S C M K A in n-propanol-water mixtures. Figure 3 shows the effect of n-propanol on the c.d. spectrum of S C M K A in aqueous solution. Because of the
insolubility of S C M K A in n-propanol, the measurements were limited to up to 90!}~,,n-propanol. In the c.d. spectra of S C M K A in n-propanol water mixtures, the magnitudes of two negative bands at 222 and 208 nm are increased slightly and decreased, respectively, in comparison with the c.d. spectrum of S C M K A in pure water. The curve-fitting analysis of the observed spectra was made with method III. The results were satisfactory except for 90% n-propanol. In Fiqure 4 is plotted the change in the content of :~-helix for S C M K A at various concentrations of n-propanol. A slight increase was observed in the a-helix content up to 50% n-propanol. However, the a-helix content remained nearly constant when the n-propanol concentration exceeded 50%, showing that the denaturing action of n-propanol on S C M K A is not so marked as in many globular proteins -~°'3~. It may be concluded that the conformation of SCMKA, even at higher concentrations of n-propanol, is little different from that in pure water, although water is removed from the environment of S C M K A molecules. Previously, Harrap 5 observed that the increase in helical content of S C M K A was small even in 2-chloroethanol, which is known to be a helix-forming solvent, and pointed out that S C M K A takes up almost its maximum helical content in aqueous solution. The results obtained here may support his conclusion. In connection with the above-mentioned results, the conformation of S C M K A in solid state was examined by c.d. The c.d. spectra of S C M K A films cast from formic acid and aqueous solution were very similar to those of S C M K A in aqueous solution, as shown in Figure 5. The spectra are expressed in terms of the difference in
5
I
I
|
I
i
I
I
( + 4 x 1 0 4)
4 #..,~
(+3 xlO 4 )
I
"6 E
3
"13
E
2
-o
l
U O1
( + 2 x 1 0 4) (+1x10 4 )
I
O "--
0
I
200
I
I
220
I
I
240
I
I
260
X Cnm) Figure 3 C.d. spectra of SCMKA in n-propanol-water mixtures (the n-propanol content in vol % is indicated). The spectra are shifted by adding the values described on each spectrum to the mean residue ellipticities in deg cm 2 drool
Int. J. Biol. Macromol., 1982, Vol 4, April
169
~-f-telical proteins l?om wool keratin: Takayuki Amiya et al.
65
I
I
i
i
SCMKA 4
.... 0-"
605
v
-
0
X
°~
0a
50'
T
45
I
I
i
i
I
10 30 50 70 90 1-Propano[(%v/v)
Figure 4 Helix content of SCMKA in n-propanol water mixtures, as a function of the n-propanol content
I
I
I
'I'
I
I
I
SCMKA
(+)
U~
r
q
,_
reversibility have been studied in detail using the Moffitt Yang parameter ho given by o.r.d, measurement 5''S C M K A has been k n o w n to undergo a thermal transition in the temperature range 25 80 C. However, the nature of the conformational change of S C M K A during thermal transition, namely, whether the conformational change of S C M K A is a helix coil or helix /~ transition, has not been elucidated. Fixture 6 shows the variation in c.d. spectra for S C M K A in water (pH 6.7) with increasing temperature. It should be noted that all the spectra intersect at 204 nm. As already mentioned, the content of fl-form is negligible in S C M K A at 20'C. Therefore, if the content of fl-form became appreciable in S C M K A at higher temperatures, the c,d. spectra would not intersect at a c o m m o n wavelength. The results of c.d. analysis by method III showed no occurrence of fi-form on heating, as can be seen in Fi,qurc 7. It may be concluded that the conformational change in S C M K A , which occurs a r r o u n d 5 0 C , is a so-called helix coil transition. In addition, the result of a visible observation should be mentioned. The S C M K A solution used for the c.d. measurements did not become turbid on heating. The turbidity of the S C M K A solution is duc to the formation of aggregates on heating and depends on the concentration 6. C.d. measurements are generally carried out with very dilute solutions. O u r observation thus suggests that no appreciable aggregation of S C M K A occurred in the concentration range employed m the present c.d. measurements (ca. 0.1 g 1 1). The results in Fi~jure 7 also show that most of the ~-helical segments are destroyed by heat, but about 15'Ii, of the helical segments are very resistant to heat. S C M K A from wool consists of a further two components, i.e. C o m p o n e n t 7 and 8 ~-~. In order to
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absorbance for left and right circularly polarized light. S C M K A in the cast films thus assumes a helical c o n f o r m a t i o n similar to that in water. The conformations of S C M K A in both cast films are almost identical, although S C M K A assumes an ~-helical or unordered c o n f o r m a t i o n in water or formic acid solution, respectively s'24. This differs from the case for the highsulphur proteins 7.
Contormational change o1 SCMK A on heatinq Thermal denaturation of S C M K A from wool and its
170
Int. J. Biol. Macromol., 1982, Vol 4, April
4O -2 .
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k (nm) Figure 6 Variation in c.d. spectra of SCMKA with increase in temperature
~-Helical proteins,l)*om wool keratin. Takayuki Amiya et al.
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6 Secondary structure of low-sulphur derivatives as estimated from c.d. analysis ".+'
protein
Table
Derivative
./n
.l}J
.11
,IR
SCMKA SAEKA
0.529 0.507
0.03 0
0.139 0.185
0.302 0.308
" Calculated by method 111.See text for details
" ./it,.l)~,,l,and.l}~are fractions of ~-helix, #-form, fi-turn and unordered form, respectively
in aqueous solution without a denaturant. In addition, preparation of these low-sulphur protein derivatives involves a number of problems -+. C.d. conformational analysis was performed on S A E K A whose protecting groups were positively charged. Table 6 shows the results of c.d. analysis of S A E K A and S C M K A . Calculations were carried out using method III under the constrained condition. Figure 8 shows the calculated c.d. spectra in water (pH 6.7) at 20 C, together with the observed spectra and the difference between the calculated and observed spectra. The results show that the charged protecting groups introduced in the low-sulphur proteins have little influence on their conformation in water. A c o m p a r i s o n of the conformation of S C M K A and esterified S C M K A was made in hexafluoro-2-propanol ( H F I P ) solution, because the esterified S C M K A was insoluble in water. S C M K A and esterified S C M K A in H F I P solution showed almost identical c.d. spectra and so did S C M K A in water. The similarity of S C M K A in water and H F I P solution suggests that S C M K A in H F I P assumes an ~z-helical conformation similar to that in pure water. However, the secondary structure contents of S C M K A and esterified S C M K A in H F I P were not estimated, since the solvent effect on protein c o n f o r m a t i o n is fairly complex 3x. F o r the sake of quantitative comparison, the mean residue ellipticities at three wavelengths characteristic of the spectra are shown in Table 7. It may be concluded that esterified S C M K A in H F I P takes approximately the same conformation as S C M K A .
5
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explore the conformational change of S C M K A in detail, a similar c.d. study of these c o m p o n e n t proteins is in progress.
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lnJluence of charged protecting groups on S C M K A conlormation In order to extract the keratin proteins from wool in a water-soluble form it is necessary to convert the cystine residues into residues with ionizable groups t 3. Most conformational studies of the keratin proteins have been carried out with SC MK, because of its superior properties such as chemical stability and high solubility in aqueous solution t 3. In the case of S C M K derivatives, however, a large number of negatively charged groups is introduced into the side chains by S-carboxymethylation, owing to the high cystine residue content in wool keratin. Therefore, it is of interest to examine the influence of these protecting groups on the c o n f o r m a t i o n of S C M K A . This section presents the results of two experiments on this subject. Low-sulphur proteins in the thiol state are unstable in the absence of reducing agent and so their c o n f o r m a t i o n cannot be investigated without a reducing agent. SAlkylated low-sulphur protein derivatives, which are protected with neutral groups, are also insoluble in water and so it is not possible to investigate their c o n f o r m a t i o n
200
220
i
240
.
i
2O0
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i
I
-3
240
X (nm) Figure 8 Comparison of the experimental and computed c.d. spectra of SCMKA and SAEKA in water tpH 6.7) at 20C. Experimental ( ); computed ( ): difference between the experimental and computed spectra (.... )
Table 7 Mean residues ellipticitics [01 of SCMKA and esterified SCMKA at three wavelengths in HFIP 'ii0] x 10 ~ (deg cm 2 dmol t) Sample
222
SCMKA Esterified
- 19.7 - 18.8
Wavelength (nm) 208 -23.7 - 22.5
191 42.2 41.8
Int. J. Biol. Macromol., 1982, Vol 4, April
171
~-Helical proteins j?om wool keratin: Takayuki Amiya et al. Last to be mentioned in this section is that the results obtained here do not reveal the influence of electrostatic charges on the conformational stability of low-sulphur proteins from wool. In order to elucidate such an influence, the denaturation temperature and pH-induced conformational transition of each derivative should be clarified. Such a study with c.d. is in progress in our laboratory. References 1 2 3 4 5 6 7 8 9 10 11
172
Bradbury, J. H. Adv. Protein Chem. 1973, 2"/, 111 Bradbury, J. H. Pure Appl. Chem. 1976, 46, 247 Crewther, W. G. in 'Proc. Int. Wool Text. Res. Conf. Aachen 1975" Vol. l, p. l Crewther, W. G., Dobb, M. G., Dowling, L. M. and Harrap, B. S. in 'Symp. on Fibrous Proteins, 1967' (Ed. W. G. Crewther} p. 329 Harrap, B. S. Aust. J. Biol. Sci. 1963, 16, 231 Harrap, B. S. Biopolymers 1969, 8, 187 Amiya, T., Miyamoto, T. and lnagaki, H. Biopolymers 1980. 19, 1093 Amiya, T., Kawaguchi, A., Miyamoto, T. and lnagaki, H. Sen-i Gakkaishi 1980, 36, 479 Greenfield, N. and Fasman, G. D. Biochemistry 1969, 8, 4108 Saxena, V. P. and Wetlaufer, D. B. Proc. Natl. Acad. Sci. USA 1971, 68, 969 Chen, Y. H., Yang, J. T. and Chau, K. H. Biochemistry 1974, 13, 3350
Int. J. Biol. Macromol., 1982, Vol 4, April
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Takahashi, S., Kontani, T., Yoneda, M. and Ooi, T. J. Biochem. 1977, 82, 1127 Chang, C. T., Wu, C. S. C. and Yang, J. T. Anal. Biochem. 1978, 91, 13 Brahms, S. and Brahms. J. J. Mol. Biol. 1980, 138, 149 Dowling, L. M. and Crewther, W. G. Prep. Biochem. 1974, 4, 203 lto, H., Miyamoto, T. and lnagaki, H. Sen-i Gakkaishi I978, 34, 157 Ito, H. unpublished experiments Crewther, W. G. and Dowling, L. M. Appl. Polym. Svmp. 1971, No. 18, 1 Holt, L. A. and Milligan, B. Aust. J. Biol. Sci. 1970, 23, 165 Baker, C. C. and lsenberg, 1. Biochemistry 1976, 15, 629 Crick, F. H. C. Acta Crystallo~]r. 1953, 6, 689 Suzuki, E., Crewther, W. G., Fraser, R. D. 8 , MacRae, T. P. and McKern, N. M. J. Mol. Biol. 1973, 73, 275 Fraser, R. D. 8 , MacRae, T. P. and Suzuki, E..1. Mol. Biol. 1976, 108, 435 Sakabe, H., Miyamoto, T. and lnagaki, H. Sei-i Gakkuishi, 1981, 37, 273 Kontani, T. J. Juzen Med. Soc. Jpn 1978, 87, 524 Parry, D. A. D., Crewther, W. G., Fraser, R. D. B. and MacRae, T. P. J. Mol. Biol. 1977, 113, 449 McLachlan, A. D. J. Mol. Biol. 1978, 124, 297 White, H. F. Jr. Biochemistry 1976, 15. 2906 Atkinson, J. C., Filson, A. and Speakman, J. B. Nature 1959, 184, 444 Bondanszky, M., Bondanszky, A., Klausner, Y. S. and Said, S. 1. Bioor~t. Chem. 1974, 3, 133 Kurono, A. and Hamaguchi, K. J. Biochem. 1964, 56, 432 Parrish, J. R. Jr. and Btout, E. R. Biopolymers 1972, 11, 1001
(Received 14 September 1981; revised 23 October 1981) The co,/brmation and col?/brmational chcmqe ~?[ wool keratin S-carboxymethylated low-sulphur proteins (SCMKA), which are ~-helieul./ibrous proteins, hal'e been int'estigated in aqueous .solution by means ~71e.d. Comparison,s ~?Jl~arious methods proposed fi)r c.d. analysis ofprotein secondary structure are made using leastsquares curt'ez/ittin,q ~?]the obsereed c.d. spectra ~?[SCMKA with a linear combination ~?[the correspomlin.q r¢~fbrence spectra ~?Jsecondary structures. It has beenJbund that (i) the most sati,~/Uctory results ure obtained with the method ' 3 which takes into account the [~-turn contribution; (ii) S C M K A is 52 54'!o cuhelical in water and has little [4:[~rm:(iii) the addition g/n-propanol produces, eeen at higher concentrations ~?[n-propanol, little change in spectra with respect to helical character in water; (iv) S C M K A undergoes a thermally-induced eo~Ibrmational tralTsition Ji'om ~-helix to random c'oil around 50 C: and (l:) S-aminoethylated low-sulphur proteins with positieely charged proteetinq ,qroups are ~ 50°0 cuhelieal in water, which is similar to S C M K A, showing that the protecting groups introduced in the low-sulphur proteins have little e[J~ct upon their confi)rmation in water. Keywords: Optical chemical analysis; keratin; fibrous protein; secondary structure; low sulphur proteins: Scarboxymethylkerateine
Introduction ~-Keratins such as wool or animal hair consist of ordered filamentous units, termed 'microfibrils' embedded in a less ordered nonfilamentous matrix l"2. Keratin proteins can be obtained as soluble derivatives after rupture of the disulphide bonds, and can be divided into three main fractions: low-sulphur, high-sulphur and high-glycine fractions 1-3. Proteins of the last two fractions do not assume a helical structure in aqueous solution and are considered to originate chiefly from the matrix. The proteins in the low-sulphur fraction, however, are partly helical in aqueous solution over a wide range of pH values, and are considered to originate from the microfibrils. Proteins from ~-keratins are studied in soluble form by reduction of the disulphide bonds and protection of the resulting thiol groups with iodoacetic acid to form Scarboxymethylkerateines (SCMK)t. Considerable progress has been made on the primary structure by chemical and physicochemical characterization of wool S C M K proteins 3. Until now, however, studies on the conformation and conformational transition of S C M K proteins in solution have relied mainly on the MoffittYang parameter bo given by o.r.d, measurement 4-6, except for our recent c.d. studies on matrix proteins 7'8. C.d. is a powerful technique for studying protein conformation in solution. When a set of reference spectra for secondary structure, a-helix, /~-form, /~-turn and unordered form are given, the content of each secondary structure in a protein can be estimated by using a leastsquares curve-fitting of the observed c.d. spectra to a * To whom correspondence should be addressed. t The term 'kerateine' is generallyused for the reduced form of keratin. 0141 8130/82/030165q38503.00 Q1982 Butterworth & Co. (Publishers) Ltd
linear combination of the corresponding reference spectra 9-14. The present paper deals with conformation and conformational change of S-carboxymethylated lowsulphur proteins (SCMKA), which are co-helical proteins from wool, in aqueous solution by c.d. The problem was divided into three objectives. The first was to find the most satisfactory set of reference spectra for the c.d. analysis of S C M K A secondary structure. Comparisons were made of three representative methods proposed for c.d. analysis of protein secondary structure 9 ' ' t t 3 The second was to investigate the conformational changes is S C M K A during alcohol and thermal denaturation. Analysis of the observed c.d. spectra was performed with the most satisfactory set of reference spectra. In the case of SCMK, a large amount of negatively charged groups is introduced into the cysteinyl residues because of the high cystine residue content of wool keratin. The third objective was to examine the influence of these charged groups on the conformation of S C M K A in aqueous solution. The conformation of S C M K A was compared with that of S-aminoethylated low-sulphur proteins (SAEKA) whose protecting groups are positively charged.
Experimental Materials
Merino 64 wool was purified according to the standard method 15. Three-times recrystallized c~-chymotrypsin from bovine pancreas was purchased from Sigma Chemical Co., USA. Deionized water was used throughout. All other chemicals were of reagent-grade and were used without further purification.
Int. J. Biol. Macromol., 1982, Vol 4, April
165
~-ftelical proteins li'om wool keratin: Tukayuki Amiya el al. Table 1 Amino acid composition ofSCMKA, SCMK-HF and SAEKA from Merino wool" Amino acid Lys His Arg AECys j' CMCys' Asp Thr Scr Glu Pro Gly Ala Cys/2 Val Met lieu Leu Tyr Phe
SCMKA SCMK-HF 3.0 0.5 6.9
5.7 0.7 7.1
7.2 8.3 5.2 9.1 16.1 4.7 8.4 6.5 0.0 4.9 0.4 3.6 8.8 3.9 2.5
3.1 11.8 3.4 6.2 20.9 1.8 2.8 8.1 6.3 0.3 4.4 14.7 0.8 1.8
SAEKA
Native
3.0 (t.5 6.9 6.9
2.9 0.~ 6,9
8.1 4.8 9.5 15.3 4.6 8.6 6.0 0.0 5.7 0.4 3.1 9.4 4.2 2.8
5.9 6.2 10.4 11.8 10.1 5.6 5.1 11.1 6.1 0.5 3.2 7.3 3.7 2.6
Values given as mol per 100 mol amino acids ~' 2-Ammoethylcysteine Carboxymetlaylcysteine
Dowling TM. The purification o f S C M K HF was repeated twice by reprecipitation at pH 4.0 from salt solution. The precipitate was dissolved in aqueous sodium borate solution. After dialysis against deionized water, the solution was freeze-dried. The molecular weight of the SCMK-H F was estimatcd by the use of a Sephadex G-75 colunm in a buffer containing 8 M urea at pH 7.4 as described previously ~' The sample consisted of three subfractions, where the major peak followed the peaks of two minor fractions of molecular weight > 2 x 104. Such an elution pattern was very similar to that for the helical fragments reported by Crewther and Dowling TM. The similarity m amino acid composition between our sample and that of Crewther et a/. was also confirmed as described later. In the present study, the S C M K - H F sample was used for the c.d. measurements without further fractionation, i.e. without removal of small amounts of higher molecular weight subfractions.
Esterflication o1 S C M K A Esterification of SCMKA was carried out by treating SCMKA with methanolic-HC1 for 24 h at 20 C ~'. The csterified SCMKA redissolved in 8 M urea solution was dialysed against deionized water and then freeze-dried.
C.d. spectra Preparation of low-sulphur protein derivatives The low-sulphur proteins of S-carboxymethylkerateine {SCM KA) were prepared from reduced wool according to the procedure of Dowling and Crewther ~s. This involved the extraction of wool with 2-mercaptoethanol at pH 10.5 in the presence of 8 M urea, the alkylation of extracted proteins with iodoacetic acid, and the precipitation of the low-sulphur proteins from 0.5 M KC1 solution at pH 4.4. The reprecipitation from salt solution was repeated three times to remove high-sulphur and high-glycine proteins. Finally, the sample fraction redissolved in sodium borate was dialysed against deonized water and then freeze-dried. The average molecular weight of SC M KA is known to be about 5 x 104 as determined by gel filtration ~' The S-aminoethylated derivative from reduced wool (SAEK) was prepared by a similar procedure to that of SCMK except that 2-bromoethylamine was used as an alkylating agent instead of iodoacetic acid ~7. The alkylation was continued by addition of 2bromoethylamine until the nitroprusside test for thiol became negative. The low-sulphur protein fraction (SAEKA) was obtained by fractional precipitation of SAEK by the procedure of Ito~ 7. The sample SAEKA was further purified by repeating precipitation from the salt solution several times to remove traces of other protein fractions. These fractionation and purification procedures were confirmed to be satisfactory from amino acid analysis and gel filtration chromatography of SAEKA on a column of Sephadex G-200 in 8 M urea ~7. Amino acid analysis also ensured that the cystinyl residue had been converted quantitatively to a 2-aminoethylcysteinyl residue and that the other residues remained unmodified Isee Table 1).
Preparation olhelix-richfi'agmentsJ?om S C M K A Helix-rich fragments (SCMK HF) from S C M K A were prepared by limited digestion of S C M K A with ~,chymotrypsin according to the method of Crewther and
166
Int. J. Biol. Macromol., 1982, Vol 4, April
C.d. spectra were taken on a Jasco J-20 spectropolarimeter in a thermostatically controlled 1 mm .jacketed cell. The c.d. data were expressed in terms of the mean residue ellipticity, [01, in deg cm 2 drool ~. The mean residue weight of each derivative was calculated from its amino acid composition. For c.d. measurements of the films, the sample solution with a different solvent was layered directly on a quartz disc used for the measurement to prepare the film. C.d. spectra were expressed in terms of the difference in absorbance for left and right circularly polarized light. The protein concentration was determined by weight since we had a sufficient quantity to prepare a stock solution of known concentration.
Amino acid analysis The sample proteins were hydrolysed in racuo at 110 C for 24 h with 6 N HCI and 2 mM phenol. The amino acid content of the hydrolysates was estimated using a Hitachi KLA-5 amino acid amdyser. The results were expressed in terms of tool per 100 tool amino acid. Table 1 summarizes the amino acid compositions of SCMKA, SCMK-HF, SAEKA and native Merino wool.
C.d. analysis In general, the c.d. spectrum of a protein is expressed m terms of a linear combination of the respective secondary structures contained in the protein. Thus the mean residue ellipticity, [0]~, at an arbitrary wavelength, 2, is given by:
[o]~. =
~ } F o ] ~..j
t l)
i where [0] ,..j denotes the reference values for the secondary structure of j, and./i its corresponding content. When a set of reference spectra for secondary structures are given, the content Ji of each secondary structure in a protein is
~-Helical proteins from wool keratin: Takayuki Amiya et ul. estimated according to equation (1) so as to yield the bestfit linear combination of reference spectra with respect to the experimental c.d. spectrum. Several methods have been proposed to analyse the spectrum 9- ~4, where the difference is due to the choice of reference spectra for each secondary structure. In the present study, c.d. analysis of S C M K A secondary structure was performed by following three representative methods. The first method (method I), which may be called the Greenfield Fasman method 9, uses the c.d. spectra of synthetic polypeptides in s-helix, //-form and random form, as reference spectra. The second (method II) is that ofChen et al. ~~ in which the calculated c.d. spectra of each secondary structure of certain proteins whose structures were estimated by X-ray data, are used as reference spectra of three structural elements. In these two methods, it is assumed that proteins contain only two types of regular structural elements: c~-helix and//-form. The third method tested (method III), is that of Yang and coworker in which the//-turn contribution is taken into account 13. Here the reference spectra (data with a 1 nm interval) of the a-helix,/#form, fl-turn and unordered form, were kindly provided by Prof. J. T. Yang. For j) determination, the following two constraints were introduced: (1) the sum of the contents of all structural elements is unity, i.e. YJi = 1, and (2) the content of each structural element is positive, i.e.Ji/> 0. The fitting was performed on a computer F a c o m M160AD by a least-squares method with 1 nm intervals, in the region 190 to 240 rim. The goodness of fit between experimental and calculated values was evaluated in terms of the root mean square error (STD) as: STD = [Z(A0~.)2/(N - i)] 1,2
c.d. spectrum characteristic of a-helix, having a positive band at 192 nm and two negative bands at 209 and 222 nm. It is characteristic of a-keratin that the low-sulphur protein fraction is largely a-helical and that its helical content is relatively high compared with that of globular proteins. The S C M K - H F sample shows a similar c.d. spectrum, but it is clear from the magnitude of the negative ellipticity bands at 209 and 222 nm that S C M K H F contains a higher content of a-helix than SCMKA. Average number of residues per helix The following two factors should be taken into consideration when estimating the secondary structure of a-keratin proteins: (1) c~-keratin consists of a characteristic coiled-coil a-helical structure 2~, and (2) the reference c.d. spectra of a-helix in methods II and III depend on the average number of residues per helix 11"13 X-ray diffraction studies on S C M K A and its helical fragments from wool indicate that these proteins consist mostly of a coiled-coil a-helical structure in the solid state22 24, while analyses of the primary structure for the helical fragments have revealed a repetitive pattern of the type expected from a-helical proteins with a coiled-coil structure a'23. We may predict that S C M K A and S C M K H F might assume a similar coiled-coil a-helical structure in aqueous solution3'% and if so the difference in the c.d. spectrum between a-helix and coiled-coil a-helix should be examined. Recently, Kontani 2s has calculated the theoretical c.d. spectra of these two a-helices, and showed that the c.d. spectrum of coiled-coil a-helix differs little from that of a-helix. This means that the reference spectra of a-helix may be used in the estimation of secondary structure of proteins containing a coiled-coil a-helix.
(2)
where A0;. is the difference between the experimental and calculated mean residue ellipticity at wavelength ), N the number of experimental data, and i the number of reference spectra used for the fitting (i.e. 3 or 4 in the present cases). Since wavelengths were taken with 1 nm intervals from 190 to 240 nm, N = 5 1 . In addition to the 'constrained' method mentioned above, the computation was also per,formed by an 'unconstrained' least-squares method 2°, i.e. without Eli = 1, but with .]j/> 0. The results of analyses using the unconstrained method provide a simple criterion for the validity of a set of reference spectra employed, because the goodness of fit can be evaluated in terms of STD as well as the deviation of Zli from unity according to Baker and lsen berg ~4,2 o.
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Results and discussion X
C.d. spectra of S C M K A and S C M K - H F in aqueous .solution S-Carboxymethylated low-sulphur proteins from wool keratin (SCMKA) have an isoelectric point at pH 4.4 and are soluble in aqueous solution when the pH value exceeds 4.4. Fiyure 1 shows the c.d. spectrum of S C M K A in aqueous solution at pH 6.7, ,together with the c.d. spectrum of helical fragments ( S C M K - H F ) prepared by partial proteolysis of S C M K A with a-chymotrypsin. The spectra of these proteins in aqueous solution were almost independent of pH in the range 6- 10. S C M K A shows a
- 2
-~1
I
200
220
!
I
240
I
260
X (rim) Figure 1 C.d. spectra of SCMKA (
) and SCMK-HF
( • - ) in water at pH 6.7
Int. J. Biol. Macromol., 1982, Vol 4, April
167
:~-Helical proteins.t?om wool keratin: Takayuki Amiya et al. Comparison ql" methods Table 4 shows the results of c.d. analysis o b t a i n e d for
The reference value of ~-helix in m e t h o d s I1 and 1II d e p e n d s on the average n u m b e r of residues per helix, n, t h r o u g h the following empirical equation9:
[0];~,= [0J,~(l
k/~0
S C M K A by the three m e t h o d s under the u n c o n s t r a i n e d condition. The calculations were carried out with h = 90. In the case of m e t h o d I, h is effectively infinite. The s u p e r i o r i t y of each m e t h o d has been discussed on the basis of the g o o d n e s s of fit between the e x p e r i m e n t a l and calculated c.d. spectra for S C M K A . The most satisfactory results were o b t a i n e d with m e t h o d I I l where a protein was a s s u m e d to c o n t a i n three types of regular structural elements including fl-turn. In Table 5 are s u m m a r i z e d results of the c.d. analysis of SCMKA under c o n s t r a i n e d conditions. Fiqure d e m o n s t r a t e s e x p e r i m e n t a l as well as calculated c.d. spectra with the respective sets of reference spectra. M e t h o d l l I gives the best fit to the observed c.d. spectrum of S C M K A . T h u s the s e c o n d a r y structure of S C M K A in a q u e o u s solution was estimated to be c o m p o s e d of 53'~,, c~helix, 3'}i, fi-form, 1470 fl-turn and 300o u n o r d e r e d form. The same calculation p r o c e d u r e was applied to S C M K H F and the s e c o n d a r y structure was found to be c o m p o s e d of 79% e-helix, 0'?~,fi-form, 171, fi-turn and 20,,, u n o r d e r e d form. Table 5 also shows that the helix content of S C M K A is almost i n d e p e n d e n t of the n a t u r e of the reference spectra chosen. Previously, H a r r a p 6 estimated the helix content of S C M K A from wool and its helical fragments using the Moffitt Yang p a r a m e t e r b o of synthetic p o l y p e p t i d e s as reference. The values of c~-helix c o n t e n t of S C M K A and S C M K - H F by the present c.d. analysis are very close to
(3)
where the superscript ,~,c,refers to a helix of infinite length and k is a chain-length i n d e p e n d e n t p a r a m e t e r (3.5 > k >2.5). The average n u m b e r h in g l o b u l a r p r o t e i n s has been k n o w n to be ~ 9 1 0 ( R e f s l l a n d l 3 ) . l n t h e c a s e of w o o l keratin, recent studies on the helical fragments from S C M K A have suggested that the p o t e n t i a l l y helical units of S C M K A would have c o n s i d e r a b l y larger h than g l o b u l a r proteins 23'2<27. However, we have no conclusive figure for n of the true helical units of SC M K A in a q u e o u s solution 3. In the present c.d. study, it was necessary to examine n for S C M K A and S C M K - H F from wool. W e have tried to evaluate n for S C M K A and S C M K - H E from the value of [( ] , which gives a best fit to the o b s e r v e d c.d. spectra. F o r this purpose, the s a m p l e S C M K - H F , which originates from the helical region of S C M K A , was used and c o m p a r i s o n s were m a d e with different fi values, with different reference values of ~-helix. It should be m e n t i o n e d here that the n - d e p e n d e n t term c o n t r i b u t e s < 5 , to the total [()]h value when h > 5 0 , as can be seen in e q u a t i o n (3). Tables 2 and 3 show the results of c.d. analysis of S C M K - H F with different h values by m e t h o d s lI a n d lII, respectively. The calculations were carried out u n d e r the u n c o n s t r a i n e d condition. A best calculated s p e c t r u m should be the one where the value of Z/i is nearly equal to unity a n d the value of S T D is the least. The results showed no significant difference in the values of S T D between the n values. However, the other criterion, i.e. ZI}, implied that the value of n should be far larger than 9. The same conclusion was also d r a w n from the calculated spectra of S C M K - H F under the c o n s t r a i n e d condition. T h u s the subsequent c.d. analyses of S C M K and S C M K - H F were carried out with n = 90.
Table 4 C.d. analysis of SCMKA secondary structure with wtrious reference spectra under the unconstrained condition
Table 2 C.d. analysis of S C M K - H F secondary structure with different n values by method II under the unconstrained conditions"
Sample
n
.li,
./)~
.I~:
ZI~
10 "~x STD b
SCMK-HF
9 20 50 90
1.110 0.996 0.905 0.881
0.079 0 0 0
0.466 0.409 0.364 0.353
1.655 1.405 1.269 1.233
0.210 0.206 0.205 0.209
"/, See text for details
Root mean square error (deg c m
Table 3
2
.lu
.li~
.11
1 II IIl
0.523 0.538 0.524
0.181 0 0 0.132
1}~
El i
10 ~ x STD"
0.112 0.330 0.280
0.816 0.868 0.936
0.176 0.103 0.069
" Root mean square error (deg cm2 dmol 1), defined in text Table 5 C.d. analysis of SCMKA secondary structure with various reference spectra under the constrained condition
Method
.fiJ
./)~
1 ll Ill
0.495 0.540 0.529
0.318 0.100 0.030
dmol L), defined in text
.11
.I~
10 4 × STD"
0.139
0.187 0.360 0.302
0.187 0.136 0.070
Root mean square error (deg cm 2 dmol t), defined in text
C.d. analysis of S C M K - H F secondary structure with different h values by method I11 under the unconstrained condition"
Sample SC M K-H F
n
.Iit
.I),
.I,
/~
Z l)
10 ~ x STD"
9
1.131
1)
0.251
0.417
1.799
0.135
20
0.979
0
0.173
0.367
1.519
0.138
50
0.890
0
0.153
0.330
1.373
0.140
90
0.866
0
0.146
0.321
1.333
0.13(/
See text for details ~' Root mean square error (deg cm 2 drool ~), defined in text
168
Method
Int. J. Biol. M a c r o m o l . , 1982, Vol 4, April
2-Helical proteins fi'om wool keratin: Takayuki Amiya et al. a
b-
#"
F~
Method Ill- ~
-6
3
i o
-2 L , 200
220
2aO
200
22-0
2L.O
200
22G
"2c.0
,
3
X(nm)
Figure 2 Comparison of the experimental and computed c.d. spectra of SCMKA in water at pH 6.7. Experimental ( -1; computed ( ): difference between the experimental and computed spectra (.... ). (a) method I; (b) method II; (c) method III those found by Harrap. Such an agreement may be attributed to the fact that the helix part of low-sulphur wool proteins is lengthy. However, it is clear that the reference spectra of method I based on synthetic polypeptides tend to considerably overestimate the Ifform content as much as 32°0, whereas other reference spectra based on globular proteins yield values < 10!?i,. Consequently, the content of unordered form was 19% with method I and ~30'!,, with other methods. In the case of reduced lysozyme 28, more satisfactory results were obtained with the reference spectra of poly(klysine) rather than those based on globular proteins. It is of interest to note that the opposite was true for reduced wool keratin although it is supposed that, unlike globular proteins, the long helix of the low-sulphur proteins from wool has an extended structure so that their shape is more like synthetic polypeptides rather than globular proteins. A great advantage of method Ill is that inclusion of the /~-turn term may improve the estimates of the/Lform and does not affect the estimates of the c~-helix~3. The present results indicate that c.d. analysis by method III is also valid for c~-helical proteins from woo1 and can explain the observed changes in their conformation in terms of the ehelix and /~-form. With regard to the /#turn, however, Chang et al. ~3 pointed out that the calculated contents were not correlated with X-ray results even for the globular proteins. This might be due to the fl-turn reference spectrum chosen. The reference spectrum by Chang et al. was not experimentally measured but extracted from different proteins and is very different from the spectra of/?-turns reported recently by Brahms and Brahms ~4. Thus the /?-turn content estimated for S C M K A would also be uncertain. However, the uncertainty of the calculated /?-turn content does not interfere with the study of the conformation and conformational changes of S C M K A in terms of the ~helix and/~-form.
SC M KA conJormation in alcoho~water mixtures Aqueous alcohol solutions, especially aqueous npropanol, are known to cause marked swelling of wool fibre 29. It is of interest to examine the conformation of low-sulphur proteins in alcohol-water mixtures. This section deals with the effect of n-propanol on the conformation of S C M K A in n-propanol-water mixtures. Figure 3 shows the effect of n-propanol on the c.d. spectrum of S C M K A in aqueous solution. Because of the
insolubility of S C M K A in n-propanol, the measurements were limited to up to 90!}~,,n-propanol. In the c.d. spectra of S C M K A in n-propanol water mixtures, the magnitudes of two negative bands at 222 and 208 nm are increased slightly and decreased, respectively, in comparison with the c.d. spectrum of S C M K A in pure water. The curve-fitting analysis of the observed spectra was made with method III. The results were satisfactory except for 90% n-propanol. In Fiqure 4 is plotted the change in the content of :~-helix for S C M K A at various concentrations of n-propanol. A slight increase was observed in the a-helix content up to 50% n-propanol. However, the a-helix content remained nearly constant when the n-propanol concentration exceeded 50%, showing that the denaturing action of n-propanol on S C M K A is not so marked as in many globular proteins -~°'3~. It may be concluded that the conformation of SCMKA, even at higher concentrations of n-propanol, is little different from that in pure water, although water is removed from the environment of S C M K A molecules. Previously, Harrap 5 observed that the increase in helical content of S C M K A was small even in 2-chloroethanol, which is known to be a helix-forming solvent, and pointed out that S C M K A takes up almost its maximum helical content in aqueous solution. The results obtained here may support his conclusion. In connection with the above-mentioned results, the conformation of S C M K A in solid state was examined by c.d. The c.d. spectra of S C M K A films cast from formic acid and aqueous solution were very similar to those of S C M K A in aqueous solution, as shown in Figure 5. The spectra are expressed in terms of the difference in
5
I
I
|
I
i
I
I
( + 4 x 1 0 4)
4 #..,~
(+3 xlO 4 )
I
"6 E
3
"13
E
2
-o
l
U O1
( + 2 x 1 0 4) (+1x10 4 )
I
O "--
0
I
200
I
I
220
I
I
240
I
I
260
X Cnm) Figure 3 C.d. spectra of SCMKA in n-propanol-water mixtures (the n-propanol content in vol % is indicated). The spectra are shifted by adding the values described on each spectrum to the mean residue ellipticities in deg cm 2 drool
Int. J. Biol. Macromol., 1982, Vol 4, April
169
~-f-telical proteins l?om wool keratin: Takayuki Amiya et al.
65
I
I
i
i
SCMKA 4
.... 0-"
605
v
-
0
X
°~
0a
50'
T
45
I
I
i
i
I
10 30 50 70 90 1-Propano[(%v/v)
Figure 4 Helix content of SCMKA in n-propanol water mixtures, as a function of the n-propanol content
I
I
I
'I'
I
I
I
SCMKA
(+)
U~
r
q
,_
reversibility have been studied in detail using the Moffitt Yang parameter ho given by o.r.d, measurement 5''S C M K A has been k n o w n to undergo a thermal transition in the temperature range 25 80 C. However, the nature of the conformational change of S C M K A during thermal transition, namely, whether the conformational change of S C M K A is a helix coil or helix /~ transition, has not been elucidated. Fixture 6 shows the variation in c.d. spectra for S C M K A in water (pH 6.7) with increasing temperature. It should be noted that all the spectra intersect at 204 nm. As already mentioned, the content of fl-form is negligible in S C M K A at 20'C. Therefore, if the content of fl-form became appreciable in S C M K A at higher temperatures, the c,d. spectra would not intersect at a c o m m o n wavelength. The results of c.d. analysis by method III showed no occurrence of fi-form on heating, as can be seen in Fi,qurc 7. It may be concluded that the conformational change in S C M K A , which occurs a r r o u n d 5 0 C , is a so-called helix coil transition. In addition, the result of a visible observation should be mentioned. The S C M K A solution used for the c.d. measurements did not become turbid on heating. The turbidity of the S C M K A solution is duc to the formation of aggregates on heating and depends on the concentration 6. C.d. measurements are generally carried out with very dilute solutions. O u r observation thus suggests that no appreciable aggregation of S C M K A occurred in the concentration range employed m the present c.d. measurements (ca. 0.1 g 1 1). The results in Fi~jure 7 also show that most of the ~-helical segments are destroyed by heat, but about 15'Ii, of the helical segments are very resistant to heat. S C M K A from wool consists of a further two components, i.e. C o m p o n e n t 7 and 8 ~-~. In order to
H2OOH
t..
n~
4
e"
i
I
SCMKA
ED (b qO
.y
v
(I)
o
(-)
E
"o
I
I
200
I
I
220
I
I
E u
I
240
2
o'1
260
1:}
;k (nm)
v
I
Figure 5 C.d. spectra of SCMKA films cast from formic acid ( - • ) and aqueous solution ( )
o,¢,-.
0
X
absorbance for left and right circularly polarized light. S C M K A in the cast films thus assumes a helical c o n f o r m a t i o n similar to that in water. The conformations of S C M K A in both cast films are almost identical, although S C M K A assumes an ~-helical or unordered c o n f o r m a t i o n in water or formic acid solution, respectively s'24. This differs from the case for the highsulphur proteins 7.
Contormational change o1 SCMK A on heatinq Thermal denaturation of S C M K A from wool and its
170
Int. J. Biol. Macromol., 1982, Vol 4, April
4O -2 .
l
I
200
|
I
220
!
i
240
k (nm) Figure 6 Variation in c.d. spectra of SCMKA with increase in temperature
~-Helical proteins,l)*om wool keratin. Takayuki Amiya et al.
¢~ 0.6
I
I,--
I
I
I
I
I
I
I
U I,.. ..I,-p
,.0 "IJ
0.4
0
u
1/1
o
0.2
¢-
cO
13-form
0
0.0
A
w
I
I
20
V
W
W
I
I
i
40
v
--
I
60
I
I
80
T (°C) Variation in SCMKA secondary structure with increase in temperature. ( © ) c~-helix; ( • ) fi-form Figure
7
6 Secondary structure of low-sulphur derivatives as estimated from c.d. analysis ".+'
protein
Table
Derivative
./n
.l}J
.11
,IR
SCMKA SAEKA
0.529 0.507
0.03 0
0.139 0.185
0.302 0.308
" Calculated by method 111.See text for details
" ./it,.l)~,,l,and.l}~are fractions of ~-helix, #-form, fi-turn and unordered form, respectively
in aqueous solution without a denaturant. In addition, preparation of these low-sulphur protein derivatives involves a number of problems -+. C.d. conformational analysis was performed on S A E K A whose protecting groups were positively charged. Table 6 shows the results of c.d. analysis of S A E K A and S C M K A . Calculations were carried out using method III under the constrained condition. Figure 8 shows the calculated c.d. spectra in water (pH 6.7) at 20 C, together with the observed spectra and the difference between the calculated and observed spectra. The results show that the charged protecting groups introduced in the low-sulphur proteins have little influence on their conformation in water. A c o m p a r i s o n of the conformation of S C M K A and esterified S C M K A was made in hexafluoro-2-propanol ( H F I P ) solution, because the esterified S C M K A was insoluble in water. S C M K A and esterified S C M K A in H F I P solution showed almost identical c.d. spectra and so did S C M K A in water. The similarity of S C M K A in water and H F I P solution suggests that S C M K A in H F I P assumes an ~z-helical conformation similar to that in pure water. However, the secondary structure contents of S C M K A and esterified S C M K A in H F I P were not estimated, since the solvent effect on protein c o n f o r m a t i o n is fairly complex 3x. F o r the sake of quantitative comparison, the mean residue ellipticities at three wavelengths characteristic of the spectra are shown in Table 7. It may be concluded that esterified S C M K A in H F I P takes approximately the same conformation as S C M K A .
5
i
,
I
,
i
,
i
,
i
,
i
,
]
I
'
i
'
i
,
i
•
SCMKA
i
'
I
SAEKA
4
o
3
%
2
E
u
I
,
explore the conformational change of S C M K A in detail, a similar c.d. study of these c o m p o n e n t proteins is in progress.
"O I
,
I
,
,
i
i
i
,
I
i
1 ........
i
0
lnJluence of charged protecting groups on S C M K A conlormation In order to extract the keratin proteins from wool in a water-soluble form it is necessary to convert the cystine residues into residues with ionizable groups t 3. Most conformational studies of the keratin proteins have been carried out with SC MK, because of its superior properties such as chemical stability and high solubility in aqueous solution t 3. In the case of S C M K derivatives, however, a large number of negatively charged groups is introduced into the side chains by S-carboxymethylation, owing to the high cystine residue content in wool keratin. Therefore, it is of interest to examine the influence of these protecting groups on the c o n f o r m a t i o n of S C M K A . This section presents the results of two experiments on this subject. Low-sulphur proteins in the thiol state are unstable in the absence of reducing agent and so their c o n f o r m a t i o n cannot be investigated without a reducing agent. SAlkylated low-sulphur protein derivatives, which are protected with neutral groups, are also insoluble in water and so it is not possible to investigate their c o n f o r m a t i o n
200
220
i
240
.
i
2O0
22O
I
i
I
-3
240
X (nm) Figure 8 Comparison of the experimental and computed c.d. spectra of SCMKA and SAEKA in water tpH 6.7) at 20C. Experimental ( ); computed ( ): difference between the experimental and computed spectra (.... )
Table 7 Mean residues ellipticitics [01 of SCMKA and esterified SCMKA at three wavelengths in HFIP 'ii0] x 10 ~ (deg cm 2 dmol t) Sample
222
SCMKA Esterified
- 19.7 - 18.8
Wavelength (nm) 208 -23.7 - 22.5
191 42.2 41.8
Int. J. Biol. Macromol., 1982, Vol 4, April
171
~-Helical proteins j?om wool keratin: Takayuki Amiya et al. Last to be mentioned in this section is that the results obtained here do not reveal the influence of electrostatic charges on the conformational stability of low-sulphur proteins from wool. In order to elucidate such an influence, the denaturation temperature and pH-induced conformational transition of each derivative should be clarified. Such a study with c.d. is in progress in our laboratory. References 1 2 3 4 5 6 7 8 9 10 11
172
Bradbury, J. H. Adv. Protein Chem. 1973, 2"/, 111 Bradbury, J. H. Pure Appl. Chem. 1976, 46, 247 Crewther, W. G. in 'Proc. Int. Wool Text. Res. Conf. Aachen 1975" Vol. l, p. l Crewther, W. G., Dobb, M. G., Dowling, L. M. and Harrap, B. S. in 'Symp. on Fibrous Proteins, 1967' (Ed. W. G. Crewther} p. 329 Harrap, B. S. Aust. J. Biol. Sci. 1963, 16, 231 Harrap, B. S. Biopolymers 1969, 8, 187 Amiya, T., Miyamoto, T. and lnagaki, H. Biopolymers 1980. 19, 1093 Amiya, T., Kawaguchi, A., Miyamoto, T. and lnagaki, H. Sen-i Gakkaishi 1980, 36, 479 Greenfield, N. and Fasman, G. D. Biochemistry 1969, 8, 4108 Saxena, V. P. and Wetlaufer, D. B. Proc. Natl. Acad. Sci. USA 1971, 68, 969 Chen, Y. H., Yang, J. T. and Chau, K. H. Biochemistry 1974, 13, 3350
Int. J. Biol. Macromol., 1982, Vol 4, April
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Takahashi, S., Kontani, T., Yoneda, M. and Ooi, T. J. Biochem. 1977, 82, 1127 Chang, C. T., Wu, C. S. C. and Yang, J. T. Anal. Biochem. 1978, 91, 13 Brahms, S. and Brahms. J. J. Mol. Biol. 1980, 138, 149 Dowling, L. M. and Crewther, W. G. Prep. Biochem. 1974, 4, 203 lto, H., Miyamoto, T. and lnagaki, H. Sen-i Gakkaishi I978, 34, 157 Ito, H. unpublished experiments Crewther, W. G. and Dowling, L. M. Appl. Polym. Svmp. 1971, No. 18, 1 Holt, L. A. and Milligan, B. Aust. J. Biol. Sci. 1970, 23, 165 Baker, C. C. and lsenberg, 1. Biochemistry 1976, 15, 629 Crick, F. H. C. Acta Crystallo~]r. 1953, 6, 689 Suzuki, E., Crewther, W. G., Fraser, R. D. 8 , MacRae, T. P. and McKern, N. M. J. Mol. Biol. 1973, 73, 275 Fraser, R. D. 8 , MacRae, T. P. and Suzuki, E..1. Mol. Biol. 1976, 108, 435 Sakabe, H., Miyamoto, T. and lnagaki, H. Sei-i Gakkuishi, 1981, 37, 273 Kontani, T. J. Juzen Med. Soc. Jpn 1978, 87, 524 Parry, D. A. D., Crewther, W. G., Fraser, R. D. B. and MacRae, T. P. J. Mol. Biol. 1977, 113, 449 McLachlan, A. D. J. Mol. Biol. 1978, 124, 297 White, H. F. Jr. Biochemistry 1976, 15. 2906 Atkinson, J. C., Filson, A. and Speakman, J. B. Nature 1959, 184, 444 Bondanszky, M., Bondanszky, A., Klausner, Y. S. and Said, S. 1. Bioor~t. Chem. 1974, 3, 133 Kurono, A. and Hamaguchi, K. J. Biochem. 1964, 56, 432 Parrish, J. R. Jr. and Btout, E. R. Biopolymers 1972, 11, 1001