Macromolecules 1990, 23, 83-88
Registry No. SPS, 28325-75-9; IPS, 25086-18-4; APS, 900353-6.
References and Notes (1) Ishihara, N.; Seimiya, T.; Kuramoto, M.; Uoi, M. Macromol-
ecules 1986,19,2464. (2) Natta, G.; Danusso, F.; Moraglio, G. Makromol. Chem. 1958, 28, 166. (3) Natta, G.; Corradini, P.; Bassi, I. W. Nuovo Cimento, Suppl. 1960,15,68. (4) Kobayashi, M.; Nakaoki, T.; Uoi, M. Rep. Progr. Polym. Phys. Jpn. 1988,31,481; Polym. Prepr. Jpn. 1988,37,2552. (5) Immirzi, A.; de Candia, F.; Iannelli, P.; Zambelli, A.; Vittoria, V. Makromol Chem., Rapid Commun. 1988,9,761. (6) Vittoria, V.; de Candia, F.; Iannelli, P.; Immirzi, A. Makromol. Chem., Rapid Commun. 1988,9,765. (7) Kobayashi, M.; Nakaoki, T.; Ishihara, N., to be published in Macromolecules. (8) Shimane, Y.; Ishioka, T.; Chatani, Y.; Ijitsu, T. Polym. Prepr. Jpn. 1988,37,2534. (9) Natta, G.; Pasquon, I.; Corradini, P.; Peraldo, M.; Pegorano, M.; Zambelli, A. Atti Accad. Naz. Lincei, C1. Sci. Fis., Mat. Nat., Rend. 1960,28,539. (10) Natta, G.; Peraldo, M.; Allegra, G. Makromol. Chem. 1964, 75,215. (11) Tadokoro, H.; Kobayashi, M.; Kobayashi, S.; Yasufuku, K.;
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Mori, K. Rep. Progr. Polym. Phys. Jpn. 1966,9,181. (12) Kobavashi. M.: Akita, K.; Tadokoro, H. Makromol. Chem. 1968,-113, 324. (13) Jasse, B.; Koenig, J. L. J . Polym. Sci., Polym. Phys. Ed. 1979, 17,799. (14) Jasse, B.; Chao, R. S.; Koenig, J. L. J. Raman Spectrosc. 1979,8, 244. (15) Girolamo, M.; Keller, A.; Miyasaka, K.; Overbergh, N. J. Polym. Sci., Polym. Phys. Ed. 1976,14,39. (16) Atkins. E. D. T.; Issac. D. H.: Keller. A,: Mivasaka. K. J . Polym.' Sci., Polym. Phys. Ed. i977,15; 21i. (17) Atkins, E. D. T.; Issac, D. H.; Keller, A. J . Polym. Sci., Polym. Phys. Ed. 1980,18,71. (18) Painter, P. C.; Kessler, R. E.; Snyder, R. W. J . Polym. Sci., Polym. Phys. Ed. 1980,18,723. (19) , . Kobavashi. M.: Tsumura. K.: Tadokoro. H. J . Polvm. Sci.. Poly;. Phys. Ed. 1968,6,' 1493. (20) Schaufelle, R. F. J . Chem. Phys. 1968,49,4168. (21) Snyder, R. G. J. Chem. Phys. 1982,76,3921. (22) Snyder, R. G.; Schlotter, N. E.; Alamo, R.; Mandelkern, L. Macromolecules 1986,19,621. (23) Snyder, R. G.; Wunder, S. L. Macromolecules 1986,19,496. (24) Snyder, R. G.; Krause, S. J.; Scherer, J. R. J. Polym. Sci., Polvm. Phvs. Ed. 1978. 16. 1593. (25) Perildo, M.;Cambini, M . 'Spectrochim. Acta 1965,21, 1509. (26) Schachtschneider, J. H.; Snyder, R. G. Spectrochim. Acta 1965,21,1527.
Dynamic Features of Side Chains in Tyrosine and Serine Residues of Some Polypeptides and Fibroins in the Solid As Studied by High-Resolution Solid-state 13C NMR Spectroscopy Hazime SaitB,*,tMika Ishida,t Motoko Yokoi,t and Tetsuo Asakuraf Biophysics Division, National Cancer Center Research Institute, Tsukiji 5-chome, Chuo-ku, Tokyo 104,Japan, and Faculty of Technology, Tokyo University of Agriculture and Technology, Nakamachi 2-chome, Koganei, Tokyo, Japan. Received November 29,1988; Revised Manuscript Received April 5, 1989
ABSTRACT We have recorded high-resolution 13C NMR spectra of silk fibroins in the solid state from Bombyx mori and Philosamia Cynthia ricini and appropriate model peptides and polypeptides to gain insight into the dynamic features of side-chain groups of Tyr and Ser residues. First, 13C NMR peaks of Tyr residues were identified, except for the peak of C , by comparing 13C NMR peaks of the B. mori fibroin with those of the crystalline fraction whose Tyr resiiue is reduced to less than 2%. It turned out that the phenolic ring of Tyr residues undergoes a flip-flop motion with a rate constant of >lo2 s-', as inferred from the coalesced single peaks of Tyr C,. The aromatic side chains of (Tyr), and (Phe),, however, undergo rotational diffusion with a correlation time on the order of lo-* s, as manifested from selective reduction of the 13C spin-lattice relaxation time of the laboratory frame (Tic). The shortened TICvalues (e1s) of Ala C, in (Ala), and fibroins were ascribed to the presence of C, rotation. The TICof Ser C, in (Ser), and fibroins appears to arise from two different relaxation processes, a shorter (0.6-1 s) and a longer (10-40 s) component. The former and latter were obviously ascribed to the presence of free hydroxymethyl groups and those hydrogen bonded to either carbonyl or other hydroxyl groups, respectively. Introduction It has been demon~tratedl-~ that the 13C NMR signals of Ala, Gly, and Ser residues in silk fibroins are well resolved i n the solid state, and the characteristic displacements of individual residues c a n be used as a conveni e n t means for distinction of polymorphic structures i n Bombyx mori and P h i l o s a m i a Cynthia ricini fibroins. T h i s view is based o n the fact that relative displacements of 13C chemical shifts from individual a m i n o acid residues
*
National Cancer Center Research Institute. Tokyo University of Agriculture and Technology.
0024-9297/90/2223-0083$02.50/0
in peptides, polypeptides, and proteins are in m a n y instances independent of amino acid sequence and significantly v a r y ( u p to 8 p p m ) w i t h the local conformation of the a m i n o acid residue under c ~ n s i d e r a t i o n . ~ - l ~ The m a j o r a d v a n t a g e in u s i n g t h i s s o l i d - s t a t e NMR approach for conformational characterization is that NMR spectroscopy is a nondestructive means and can be equally applied t o s y s t e m s of noncrystalline as well as crystalline samples. To m a k e t h i s
NMR methodology more effective, i t is essential to resolve and assign as m a n y 13C NMR signals as possible. No a s s i g n m e n t of peaks, however, has been 0 1990 American Chemical Society
84 SaitG et al.
made for the Tyr residue, despite the fact that Tyr plays an important role as an active site in the development of the silk fibroin fiber with a covalently immobilized enzymel'*l* and also as a reaction site with the azo dyes such as Orange 11.'' This is because its relative proportion is low (4.8%),' as compared with those of Ala, Gly, and Ser residues in B. mori fibroin, and many of its aromatic peaks were overlapped with the spinning sidebands of the carbonyl peaks. The latter contribution, however, is now easily removed by the pulse sequence of the total suppression of spinning sidebands (TOSS).'l Furthermore, this NMR approach can be extended to analyze dynamic features of silk fibroins, especially the manner of side-chain orientation or motion, either by examination of line shape or by means of relaxation parameters of NMR spectroscopy. Analysis of the molecular motion of the fibroin in the solid is very important in relation to understanding their physical state as mentioned above. T o this end, we have previously showed on the basis of an 'H NMR study of [2-Ser-'H2]- and [2-Al~-~H,]-fibroin that the majority of the Ser C, hydroxymethyl group is hydrogen bonded to either hydroxyl or carbonyl groups, whereas the C, methyl group of Ala residues undergoes rapid C rotation on the NMR time scale." Examination of the 3H NMR line shape of the powder pattern permits one to study dynamic features on the intermediate time scale of 10-5-10-6 s such as in the flip-flop motion of the phenolic ring in Tyr.23,24 Much slower motion can be detected by chemical exchange of isotropic peaks.25 On the other hand, more rapid motion such as rotational diffusion with a time scale of lO-'s can be monitored by 13C spin-lattice relaxation times of the laboratory frame. In this paper, we recorded 13C NMR spectra of a variety of fibroin samples as well as a number of model peptides and polypeptides to examine the presence or absence of flip-flop motion in the phenolic ring of Tyr residues. We further extended this NMR approach to study mainchain or side-chain motions of fibroins and some polypeptides by examination of 13C spin-lattice relaxation times (TICs)of the laboratory frame.
Experimental Section Poly(L-tyrosine)(Sigma Type 11, P-8523), poly(L-phenylalanine) (Sigma, P-8254) and poly(L-serine)(Sigma, P-5887) were used without further purification. Preparation and spectral characterization of poly(L-alanine)(PLA-200) and Z-(L-Ala),-NH(CH,),CH, were described previou~ly.~ L-Tyrosylglycine (T5254), N-acetyl-L-tyrosine ethyl ester (A-6751), N-acetyl+ tyrosinamide (A-6626), and L-tyrosinamide (T-3879) were purchased from Sigma Chemical Co. Preparation of N-acetylL-tyrosine methylamide was previously described.26 Cocoons of B. mori and P. c. ricini were obtained in our laboratory. The B. mori fibroin was degummed with 0.5% citric acid solution. The crystalline (Cp) fraction was obtained by digestion of the aqueous solution of the degummed fibroin by chymotrypsin, followed by collection of the resulting precipitate~.','~An oligopeptide model of the Cp fraction, Boc-(GlySer-Gly-Ala-Gly-Ala),-OBzl,l was a generous gift from Professor Mitsuaki Narita of Tokyo University of Agriculture and Technology. Highly crystalline silk I preparation was obtained from precipitates resulting from the dialysis of a 9 M LiBr solution of the Cp fraction against distilled water containing CH,COOH (pH
