2002 60 12 2203 2208 ACTA CHIMICA SINICA Vol 60 2002 No 12 2203 2208 : Ξ a b a KNIGHT David P b VOLLRATH Fritz b Ξ ( a 200433) ( b OX1 3PS ) NaCl > KCl > LiCl 2 KCl > LiCl > NaCl KCl ( ) ( 5 min ) ( 50 min ) Conformation Transition of Silk Protein Membranes Monitored by Time2resolved FTIR Spectroscopy : Effect of Alkali Metal Ions on Nephila Spidroin Membrane CHEN Xin Ξ a b SHAO Zheng2Zhong a KNIGHT David P b VOLLRATH Fritz b ( a Department of Macromolecular Science Key Laboratory of Molecular Engineering of Polymers of Education Ministry Fudan University Shanghai 200433) ( b Department of Zoology University of Oxford South Parks Road Oxford OX1 3PS UK) Abstract The conformation transition processes of Nephila spidroin membranes induced by alkali salt solutions were monitored by time2resolved FTIR spectroscopy Though the transition rate of spidroin in the membrane was NaCl > KCl > LiCl the 2sheet structure content in the membrane ( after the conformation transition completed) was KCl > LiCl > NaCl Comparing the transition rate and final 2sheet structure content in the membrane KCl has the best effect in inducing the conformation transition of spidroin membrane among the three alkali salts we used This supports the assumption that the K + ions play an important role in the spinning process of spiders In the meantime the conformation transition process was indicated to have two intermediate states that contain different levels of 2sheet structure The first phase (fast phase with a time constant of 5 min) was assigned to the 2sheet formation by segments movement while the second phase ( slow phase with a time constant of 50 min) was attributed to the whole macromolecular rearrangement Keywords time2resolved FTIR spectroscopy Nephila spidroin membrane conformation transition kinetics Ξ E2mail : chenx @fudan edu cn ; Fax : 021265640293 Received May 15 2002 ; revised August 2 2002 ; accepted August 28 2002 (No 29974005) Biotechnology and Biological Sciences Research Council (BBSRC) Engineering and Physical Sciences Research Council ( EPSRC)
2204 Vol 60 2002 alkali spinning mechanism K + ( dragline) 1 Kevlar [1] 1 1 [18 ] [2 4 ] ( Nephila senegalensis) ph 8 2 Ringer (major ampullate) Ringer (spidroin dope) ( ) 2 w = 2 % 1 ml 3 cm 3 cm (25 [1 5 6] [7 ] 50 % ) ( 24 h) 5 m 1 2 / ( ) 25 Nicolet Magna 550 [8 9 ] [10 11] [12] JUN2AIR Peak Scientific MCT 128 4 cm - 1 [13 ] 3 mm 6 mm 100 m 1 0 mol/l ( [14 16 (folding) ] Aldrich) (128 ( Na + K + ) 4 cm - 1 ) K + [17 ] 0 58 min K + 240 min (nanofibrils) ( difference spectra) : [18] [19 ] t K + ( 10 mmol/ L 1 0 mol/ L) (240 min ) K + t 1 0 mol/ L (Li + Na + K + ) Microcal TM Origin TM 5 0 ; PeakFit TM 4 1 ( SPSS Inc )
No 12 : 2205 2 2 1 ( ) 3a 3b 2a 2b I [13 ] : 2 ( 1 ( a) : I ) ( 1648 cm - 1 ) [20 23] [13 ] 1 0 mol/ L : 1620 1691 cm - 1 1648 cm - 1 ( 1 b) 1620 cm - 1 2 1691 cm - 1 2 (turn) [20 23 ] [19 ] 2 1691 cm - 1 ( 2b) : 2 (1620 cm - 1 ) (1691 cm - 1 ) ( ) 1 (a) (b) 2 1 0 mol/ L (a) ; (b) Figure 2 Time2resolved FTIR spectra of Nephila spidroin membranes induced by 1 0 mol/ L NaCl/ D 2 O solution (a) Normal spectra ; (b) difference spectra ( 1 0 mol/ L ) 2 2 Figure 1 FTIR spectra of Nephila spidroin membranes before (a) [13 and after (b) 19] the conformation transition occurred (induced by 1 0 mol/ L LiCl/ D 2 O solution) ( absorbance) 2a 1 0 mol/ L NaCl 4 1 0 mol/ L 2 2 (1648 cm - 1 ) 2 (1620 cm - 1 )
2206 Vol 60 2002 3 1 0 mol/ L (a) ; (b) Figure 3 Three dimensional time2resolved FTIR spectra of Nephila spidroin membranes induced by 1 0 mol/ L NaCl/ D 2 O solution (a) Normal spectra ; (b) difference spectra membrane monitored by time2resolved FTIR spectroscopy (a) Increasing of 2 ( ) 1 0 mol/ L 1 0 mol/ L ( 5) ( 2 ) (second 4 1 0 mol/ L (a) 1620 cm - 1 2 ; (b) 1648 cm - 1 Figure 4 Conformation transition kinetics of Nephila spidroin around 1648 cm - 1 2sheet at 1620 cm - 1 ; ( b) decreasing of random coil order exponential decay) 5 ( 1 2 ) (1620 cm - 1 ) ( 1 ) 1 Figure 5 Comparison of conformation transition kinetics of Nephila spidroin membranes induced by different alkali salt solutions (1620 1 ( ) ( 1620 cm - 1 52 % 1691 cm - 1 cm - 1 band) 42 %) 1 ( ) 10 % 1
No 12 : 2207 1 0 mol/ L a 1 Table 1 Conformation transition kinetics of Nephila spidroin membranes induced by alkali salt solutions 1620 cm - 1 1 / min 2 / min 1 / % 1691 cm - 1 1 / min 2 / min 1 / % 3 6 0 6 54 9 5 1 42 8 4 1 2 2 66 2 3 8 32 3 4 6 0 6 49 0 5 7 52 2 4 4 0 8 48 7 6 2 41 1 7 4 2 4 53 3 9 5 51 7 7 8 2 1 54 9 6 2 43 7 a A ( absorbance) ( 1 ) 1 1 = A 1 / (A 1 + A 2 ) A 1 A 2 1 2 2 3 2 ( ) 2 ( 5 1) [13 ] : [17 / ] ; ; (nanofibril) [18 2 ] ( ) 4 9 [24 ( ) 25] ( 24 h [26] 1 ( ) 4 h ) 2 2 2 PeakFit TM 2 2 2 [19 ] : 2 ( ) 2 2 ( ) 2 2 2 ; ; 2 2 2 ( K + / Na + [17 ) ]
2208 Vol 60 2002 2 5 Kerkam K ; Viney C ; Kaplan D ; Lombardi S Nature [7] 2 6 Willcox P J ; Gido S P ; Muller W ; Kaplan D L Macromolecules 1996 29 5106 7 Li G Y ; Zhou P ; Shao Z Z ; Xie X ; Chen X ; 2 2 Wang H H ; Chunyu L J ; Yu T Y Eur J Biochem 2001 268 6600 Table 2 2Sheet content in Nephila spidroin membranes after the 8 Lenormant H Trans Faraday Soc 1956 52 549 conformation transition induced by different alkali salts solutions 9 Hijirida D H ; Do K G ; Michal C ; Wong S ; Zax a 1 0 mol/ L 2 / % Abs 1620 / Abs 1691 D ; Jelinski L W Biophys J 1996 71 3442 18 2 3 2 3 3 0 8 10 Shao Z ; Vollrath F ; Sirichaisit J ; Young R J 17 2 2 0 2 7 0 7 Polymer 1999 40 2493 22 7 0 5 4 3 0 4 11 Shao Z Z ; Young R J ; Vollrath F Int J Biol a Abs 1620 / Abs 1691 2 (1620 Macromol 1999 24 295 cm - 1 ) (1691 cm - 1 ) ( 12 Chen X ; Li W J ; Yu T Y J Polym Sci Part B : ) 3 14 Reinstadler D ; Fabian H ; Backmann J ; Naumann D Biochemistry 1996 35 15822 15 Williams S ; Causgrove T P ; Gilmanshin R ; Fang K S ; Callender R H ; Woodruff W H ; Dyer R B 2 Biochemistry 1996 35 691 16 Gilmanshin R ; Williams S ; Callender 2 R H ; 15006 Mr Paul Embden 18 Chen X ; Knight D P ; Vollrath F Biomacromolecules 19 Chen X ; Kinght D P ; Shao Z Z ; Vollrath F Biochemistry 2002 in press References 1 Vollrath F ; Knight D P Nature 2001 410 541 2 Liivak O ; Blye A ; Shah N ; Jelinski L W Macromolecules 1998 31 2947 3 Seidel A ; Liivak O ; Jelinski L W Macromolecules 1998 31 6733 4 Seidel A ; Liivak O ; Calve S ; Adaska J ; Ji G D ; Yang Z T ; Grubb D ; Zax D B ; Jelinski L W Macromolecules 2000 33 775 1991 349 596 Polym Phys 1997 35 2293 13 Chen X ; Shao Z Z ; Marinkovic N S ; Miller L M ; Zhou P ; Chance M R Biophys Chem 2001 89 25 Woodruff W H ; Dyer R B Biochemistry 1997 36 17 Knight D P ; Vollrath F Naturwissenschaften 2001 88 179 2002 3 644 20 Byler D M ; Susi H Biopolymers 1986 25 469 21 Surewicz W K ; Mantsch H H Biochim Biophys Acta 1988 952 115 22 Dong A ; Huang P ; Caughey W S Biochemistry 1990 29 3303 23 Simonetti M ; Di Bello C Biopolymers 2001 62 95 24 Canetti M ; Seves A ; Secundo F ; Vecchio G Biopolymers 1989 28 1613 25 Simmons A H ; Michal C A ; Jelinski L W Science 1996 271 84 26 Helfand E Science 1984 226 647 (A0205152 LI L T ; FAN Y Y )