Crystal Structure of Silk of Bombyx mori - ACS ... - ACS Publications

Chapter 15 Crystal

Structure

of

Silk

o f Bombyx

mori

Yasuhiro Takahashi

Downloaded by PURDUE UNIVERSITY on September 13, 2013 | http://pubs.acs.org Publication Date: December 3, 1993 | doi: 10.1021/bk-1994-0544.ch015

Department of Macromolecular Science, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan

The crystal structure of silk fibroin (Bombyx mori) was reexamined by using newly collected intensity data. Four molecules pass through a rectangular unit cell with parameters, a = 9.38 A, b = 9.49 A, and c (fiber axis) = 6.98 A, and the space group P2 -C 2. The sheet formed by hydrogen bond is arranged parallel to the ac-plane. The molecular conformation is essentially the same pleated-sheet conformation as Marsh, Corey, and Pauling (1) proposed, but the sheet formed by hydrogen bonds assumes the antipolar-antiparallel structure in which the methyl groups of the alanine residues alternately point to both sides of the sheet along the hydrogen bonding direction, differing from the polar-antiparallel structure proposed by Marsh, Corey, and Pauling in which the methyl groups are a l l on the one side of the sheet. In the crystal structure of silk, two antipolar-antiparallel sheets with different orientations statistically occupied a crystal site with different probabilities . The crystalline region of silk is composed of rather irregular stacking of the antipolar-antiparallel sheets with different orientations. 1

2

The structural study of silk {Bombyx mori )can be said to start from the work of Nishikawa and Qno in 1913, in which they pointed out the characteristic feature of the so-called "fiber diagram" (2). In 1955, the crystal structure of silk fibroin was proposed by Marsh, Corey, and Pauling (1), which has been accepted so far. However, their crystal structure model is based on the quantitative intensity estimation of six equatorial reflections (discrepancy factor R = 37 %) and the qualitative comparison between the calculated and observed intensities on the layer lines. Judging from the present level of the crystal structure analysis of the fibrous sample, it is insufficient to be accepted. The purpose of

0097-6156/94/0544-0168$06.00/0 © 1994 American Chemical Society In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

15. TAKAHASHI

Crystal Structure of Silk ofB. mori


the p r e s e n t study i s t o reexamine t h e c r y s t a l s t r u c t u r e . r e s u l t s are p a r t l y given i n the note (3).

169 The

The cocoon f i b e r s were r o l l e d up on a metal holder i n a shape of c y l i n d e r w i t h 1 mm diameter after p u r i f i c a t i o n according to the usual procedure. The specimen used for Weissenberg photograph was prepared by c u t t i n g i t o f f 1 mm long after s t i c k i n g by Aron Alpha. The doubly oriented sample was prepared by r o l l i n g the s i l k gland from the lumen. X - r a y measurements were c a r r i e d out by CuKU r a d i a t i o n monochromatized by a p y r o l y z e d g r a p h i t e . The f i b e r diagram o f s i l k f i b r o i n i s shown i n F i g u r e 1. The i n t e n s i t i e s o f f o u r s t r o n g r e f l e c t i o n s were measured by a PSPC system. The integrated i n t e n s i t i e s of 26 r e f l e c t i o n s were measured by a drum s c a n d e n s i t o m e t e r and f i v e weak r e f l e c t i o n s were v i s u a l l y estimated, after f i b e r and Weissenberg photographs were taken by the m u l t i p l e f i l m method. Some o v e r l a p p e d r e f l e c t i o n s are s e p a r a t e d by the l e a s t - s q u a r e s method under t h e assumption o f Gaussian p r o f i l e . Thus, the i n t e n s i t i e s o f 35 independent r e f l e c t i o n s were c o l l e c t e d . The p r o t e i n o f s i l k f i b r o i n i s composed o f L - and H-chains l i n k e d by a d i s u l f i d e bond, the molecular weights of which are ca. 25,000 and ca. 350,000, respectively. The amino a c i d sequences of the L-chain and a part of the Η-chain are c l a r i f i e d ( 4 - 7 ) . It i s not considered that the L-chain i s associated w i t h the c r y s t a l l i n e region judging from the amino a c i d sequence. The regular sequence of the Η-chain i s associated w i t h the c r y s t a l l i n e region(7, 8). The amino a c i d sequence i n the c r y s t a l l i n e region i s considered as (GAGAGS-) where G, A , and S denote g l y c i n e , a l a n i n e , and s e r i n e , r e s p e c t i v e l y . ( 6 , 7) As the f i r s t a p p r o x i m a t i o n , the amino a c i d sequence i s c o n s i d e r e d t o be ( - G A - ) . The f i b e r i d e n t i t y p e r i o d was e s t i m a t e d as 6.98 A, a l t h o u g h t h e v e r y weak s t r e a k l a y e r s corresponding to the sequence (-GAGAGS-) are observed between the equator and the f i r s t layer l i n e . From the f i b e r i d e n t i t y period, the m o l e c u l a r c o n f o r m a t i o n i s c o n s i d e r e d t o be e s s e n t i a l l y the pleated-sheet structure as Marsh et a l . ( 1 ) proposed. The observed r e f l e c t i o n s can be indexed by the r e c t a n g u l a r u n i t c e l l w i t h parameters, a = 9.38 A, b = 9.49 A, and c ( f i b e r a x i s ) = 6.98 A and the space group P2-J-C9 , which are e s s e n t i a l l y the same as those reported by Marsh et al.(1) The u n i t c e l l contains four molecular chains, a p a i r of which forms a sheet structure by hydrogen bonds. From the space group P2-|, two m o l e c u l e s f o r m i n g the sheet i n the u n i t c e l l are symmetrically independent and two sheets are r e l a t e d by t w o - f o l d screw a x i s . Four models can be c o n s t r u c t e d f o r the hydrogen bonding sheet, p o l a r - a n t i p a r a l l e l sheet (PA), p o l a r p a r a l l e l sheet (PP), a n t i p o l a r - a n t i p a r a l l e l sheet (AA), a n t i p o l a r p a r a l l e l sheet (AP) models ( F i g u r e 2). The m o l e c u l e s i n the p a r a l l e l sheet models assume the same d i r e c t i o n w h i l e , i n the a n t i p a r a l l e l sheet models, the up- and down-molecules alternate. In the polar models, the methyl groups of alanine residues are a l l on one side of the sheet, while i n the a n t i p o l a r models, the methyl groups a l t e r n a t e l y p o i n t t o b o t h s i d e s o f the sheet a l o n g the hydrogen bonding d i r e c t i o n . The c r y s t a l s t r u c t u r e proposed by n

n

n

In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


170

SILK POLYMERS: MATERIALS SCIENCE AND BIOTECHNOLOGY

Figure 1.

X-ray f i b e r diagram of s i l k (Bombyx mori).


15.

TAKAHASHI


o-*£-o o ^ - o

171

Crystal Structure of Silk ofB. mori

o-^-o

o^-o

o-^-o

o-^-o

o-^-o

o-^-o

et»)

o-^Lo

o-^Lo o ^ - o

o-^Lo o ^ - o o ^ - o o-^L-o

( d ) Figure 2.

Four kinds of sheet structure models. (a) P o l a r - a n t i p a r a l l e l model which Marsh et a l (1) proposed, (b) p o l a r - p a r a l l e l model, (c) a n t i p o l a r a n t i p a r a l l e l model, and (d) a n t i p o l a r - p a r a l l e l model.


172


Marsh et a l . corresponds to the regular arrangement of the p o l a r ant i p a r a l l e i sheets (1). The constrained least-squares refinement (9, 10) was c a r r i e d out by u s i n g the accepted bond l e n g t h s and bond a n g l e s , where the same c o n f o r m a t i o n was assumed f o r the symmetrically independent molecules and the anisotropic temperature factor exp[-(ha*/2) B^ + ( k b * / 2 ) B + ( l c * / 2 ) B ) ] was adopted by t a k i n g i n t o account the a n i s o t r o p y i n v i b r a t i o n and d i s o r d e r . The r e f i n e m e n t was f i r s t made for the models based on the regular arrangement of the sheets. R - f a c t o r s converged t o 17.9 % f o r PA, 19.8 % f o r PP, 24.5 % f o r AA, and 18.3 % f o r AP. The PA model g i v e s the l o w e s t R - f a c t o r , but these values are not n e c e s s a r i l y s u f f i c i e n t t o d i s t i n g u i s h which model i s c o r r e c t . Many r e s e a r c h e r s p o i n t e d out the i n t e r - and i n t r a - s h e e t d i s o r d e r i n the c r y s t a l l i n e r e g i o n o f s i l k and the r e l a t e d p o l y p e p t i d e s (11 - 13). The anisotropy i n the r e f l e c t i o n broadening observed on X-ray d i f f r a c t i o n pattern, e s p e c i a l l y on the doubly o r i e n t e d sample, suggests t h a t the s i l k i n c l u d e s the d i s o r d e r i n the d i r e c t i o n p e r p e n d i c u l a r t o the hydrogen bonding sheets, i . e., the stacking disorder of the hydrogen bonding sheet (intersheet disorder). The disorder was taken i n t o account by the s t a t i s t i c a l c o e x i s t e n c e o f t h e f o u r sheets w i t h d i f f e r e n t orientations at a c r y s t a l s i t e with d i f f e r e n t p r o b a b i l i t i e s . This t y p e o f d i s o r d e r was f o u n d i n t h e c r y s t a l s t r u c t u r e o f p o l y ( v i n y l i d e n e f l u o r i d e ) form I I (14, 15) and n a t i v e c e l l u l o s e (16). Each sheet models are c o n s t r u c t e d by a l l o t t i n g the same p r o b a b i l i t y to the molecules forming the sheet at two symmetrically independent c r y s t a l s i t e s . The r e f i n e m e n t gave f a r b e t t e r agreement between the observed and c a l c u l a t e d structure factors: Rf a c t o r s , 13.7 % f o r PA, 13.8 % f o r PP, 9.7 % f o r AA, and 11.6 % f o r AP. AA model gave the l o w e s t R - v a l u e s 9.7 %, i n s p i t e o f the f a c t that, i n the regular arrangement, AA model gave the largest R-value 24.5 %. 2

2

2


z

F i n a l l y , s e r i n e r e s i d u e were taken i n t o c o n s i d e r a t i o n by r e p l a c i n g the 1/3 of a l a n i n e by the 1/3 o f s e r i n e i n w e i g h t . Rvalue d i d not reduce i n the cases of PA and PP models, but when the i n t e r n a l r o t a t i o n angle C(=0)-C(ot)~c( β)-Ο(Η) assumes gauche conformation, AA model gave the lowest value 8.5 %, while PA model gives 10.7 % when the bond assumes minus gauche. Therefore, i t i s concluded that the AA model i s most probable. The p r o b a b i l i t i e s of the molecules w i t h d i f f e r e n t orientations occupied a c r y s t a l s i t e are 72 %, 24 %, 1 %, and 4 %. A c c o r d i n g l y , w i t h i n the a c c u r a c y o f the p r e s e n t a n a l y s i s , i t can be s a i d t h a t a c r y s t a l s i t e i s occupied i n the r a t i o 3 : 1 by two sheets (AA) r e l a t e d by t w o - f o l d r o t a t i o n a x i s p a r a l l e l to the b - a x i s . I n o t h e r words, t h e c r y s t a l l i n e region of s i l k i s constructed by stacking of two kinds of sheets w i t h different o r i e n t a t i o n s . The c r y s t a l structure (AA model) i s shown i n Figure 3 and the molecular structure i s shown i n F i g u r e 4 i n comparison w i t h the p l e a t e d sheet model r e p o r t e d by Marsh e t a l . A sheet s t r u c t u r e (AA) formed by hydrogen bonds i s shown i n Figure 5.



TAKAHASHI

Crystal Structure of Silk of B. mori

#

1

#

a = 9.38A

Figure 3.

Crystal structure of

silk.


174


150.9° e

142.0 171. 4

e

e


134.0 154.2° e

170.6

Marsh et. al Figure 4.

Figure 5.

AntipolarAntiparallel lAA-HI)

Molecular structure of

silk.

Sheet structure formed by hydrogen bonds of s i l k . Figures are given i n A unit.


15. TAKAHASHI Crystal Structure of Silk of B. mori


Literature Cited 1. Marsh, R. E.; Corey, R. B.; Pauling, L., Biochim. biophys. Acta , 1955, 16, 1. 2. Nishikawa, S.; Ono, S., Proc. Tokyo Math. Phys. Soc., 1913, 7, 131. 3. Takahashi, Y.; Gehoh, M.; Yuzuriha, K., J. Polym. Sci. Polym. Phys. Ed., 1991, 29, 889. 4. Shimura, K., Experimentia, 1983, 39, 455. 5. Yamaguchi, K.; Kikuchi, T.; Takagi, Α.; Kikuchi, F.; Oyama, F. Shimura, K.; Mizuno, S., J. Mol. Biol. , 1989, 210, 127. 6. Tsujimoto, Y.; Suzuki, Y., Cell., 1979, 18, 591. 7. Lucas, F.; Shaw, J. T. B.; Smith, S. G., Biochem. J. , 1957, 66, 468. 8. Warwicker, J. O., Acta Cryst. , 1954, 7, 565. 9. Takahashi, Y.; Sato, T.; Tadokoro, H.; Tanaka, Y., J. Polym. Sci. Polym. Phys. Ed., 1973, 11, 233. 10. Takahashi, Y., Rep. Progr. Polym Phys. JPN. , 1992, 35, 231. 11. Arnott, S; Dover, S. D.; Elliot, Α., , 1967, 30, 201. 12. Fraser, R. D. B.; MacRae, T. P.; Parry, D. A. D.; Suzuki, E., Polymer, 1969, 10, 810. 13. Lotz, B.; Brack, Α.; Spach, G., J. Mol. Biol. , 1974, 87, 193. 14. Takahashi, Y.; Matsubara, Y.; Tadokoro, H., Macromolecules, 1983, 16, 1588. 15. Takahashi, Y; Tadokoro, H., Macromolecules , 1983, 16, 1880. 16. Takahashi, Y.; Matsunaga, H., Macromolecules, 1991, 24, 3968. RECEIVED

July 7, 1993


Crystal Structure of Silk of Bombyx mori - ACS ... - ACS Publications

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