1、General introduction about silk Silk is a kind of natural product. This material is superior to almost synthetic fibers with regard to the combination of energy to break and tensile strength. Understanding the concepts of this remarkable feature and translating it to synthetic materials can result i
2、n improved engineering polymers. 桑蚕丝素蛋白的分子量约为桑蚕丝素蛋白的分子量约为 2.5 105 3.5 105Amino acid Native B. mori silk fibroin Regenerated B. mori silk fibroin ASP/ASN 2.4 1.46 THR 1.6 0.8 SER 12.3 10.8 GLU/GLN 1.2 0.9 PRO 0.7 0.5 GLY 43.5 46.2 ALA 28.0 29.7 CYC 0.1 Not determined VAL 2.3 2.1 MET 0.1 0.1 ILE 0.6 0
3、.5 LEU 0.5 0.4 TYR 5.0 4.9 PHE 0.7 0.6 HIS 0.2 0.2 LYS 0.5 0.3 ARG 0.6 0.4 Amino acid composition (mol%) of native and regenerated by B.m. silk fibroin氨基酸残基的主要序列为：氨基酸残基的主要序列为：GAGAGS or GAGAGAGS存在至少两个非二硫键连存在至少两个非二硫键连接的亚单元接的亚单元SpidersThere are more than 35,000 kinds of spider living in this world. The
4、ir history can be trace back to 400 million years ago.Other spiders and webs Bolars spiderNetcasting Spider蜘蛛能分泌多种具有不同功能的丝蛋白蜘蛛能分泌多种具有不同功能的丝蛋白Spider silks从其主腺体从其主腺体(Major Ampullate) 中中吐出的丝是目前研究的主要对象吐出的丝是目前研究的主要对象Spider s spigots and silksAmino acid compositions of various spidroin蚕丝素蛋白纤维的蚕丝素蛋白纤维的不规则性
5、不规则性Hierarchical structure of Bombyx mori silk研究动物丝和丝蛋白的意义 丝所独具的优异力学性能 非常温和且友好的成丝过程（蛋白质的构象变化 ） 丝及其丝蛋白的环境可降解性 丝及其丝蛋白的生物相容性 丝及其丝蛋白的化学和物理可修饰性 目前即使质量最好的人目前即使质量最好的人造高分子材料，在弹性、造高分子材料，在弹性、断裂吸收能、纤维长度、断裂吸收能、纤维长度、重量比等综合力学性能上重量比等综合力学性能上依然还无法超越自然界中依然还无法超越自然界中的动物丝。的动物丝。通过对丝及其相关蛋白质的分析，建立天然生物大分子和合成高分子之间的联系，最终找到人工生
6、产具有类似特性产品的方法成丝过程的成丝过程的神奇之处神奇之处合成纤维：110 m/s液晶的形成液晶的形成蛋白质构象的转变蛋白质构象的转变Mystic spinning process of animal?动物丝：12 cm/s水不溶水不溶的纤维的纤维园二色谱园二色谱小角小角X射线衍射射线衍射偏光显微镜等偏光显微镜等拉伸仪拉伸仪拉曼光谱仪拉曼光谱仪广角广角X射线衍射射线衍射流变仪等流变仪等蛋白质的蛋白质的自组装自组装水溶性水溶性的凝胶的凝胶动物纺丝的动物纺丝的速率远低于速率远低于合成纤维合成纤维各种影响因素?各种影响因素?Silkgland of Bombyx mori. AB: Ant. di
7、vi., 35mm, 0.050.3mm ; BC: Middle divi., 65mm, 1.22.5mm ; CD: Post. divi., 220, 0.40.8mm Fiberization of B. mori silk fibroin水分挥发水分挥发拉伸应力拉伸应力高剪切应力高剪切应力蛋白质浓度增大蛋白质浓度增大有序化开始有序化开始分子链排列分子链排列分子链进一步有序排列分子链进一步有序排列12丝素水溶液，无规线团丝素水溶液，无规线团20水凝胶，水凝胶，复合螺旋复合螺旋23水凝胶，水凝胶，无规的螺旋无规的螺旋+-螺旋螺旋25水凝胶，水凝胶， -螺旋螺旋+拉开的螺旋，液晶形成拉开
8、的螺旋，液晶形成30水凝胶，水凝胶， -折叠，向列型液晶折叠，向列型液晶固体丝，高取向的固体丝，高取向的 -折叠结构折叠结构Dissected MA gland & associated structure (Nephila Edulis)a, A-zone; b, B-zone; f, funel; 1,2,3, first, second and third limbs of duct; m, duct levator muscle; v, valve; vm, valve tensor muscle; t, terminal tubule; s, spigot. Scale bar 1m
9、mThe formation of spider silk in duct(B) As A, but showing a highly shortened draw down taper and coiling of a thread in the distal part of the third limb of the duct before the valve. The shortening of the draw down taper is likely to result from stress release on rupture. Scale bar 50 m.(A) Interf
10、erence contrast micrograph of whole mount showing a helical twisting of the thread within the lumen of third limb of the duct at the start of the draw down taper in an individual with an unruptured thread. This could result from a spontaneous coiling of the thread or from the proposed twisting actio
11、n of the valve. Scale bar 50 m.States of liquid fibroin in B. mori silk glandMechanical properties of silksCocoon silk断裂能 (X10 KJ/Kg)0246810121416蜘蛛丝蚕茧丝凯夫拉纤维纤维素纤维肌腱骨高延展性钢Mechanics of single filament of spider silkSpider silk has a large recoverability after it is stretched, not only under normal con
12、dition, but in the selected solventsStress-strain curves of Araneus d. dragline during (wet state) and after(re-dried state) contraction in the liquids. Shortage was defined as negative strainOrientation of -sheet in silksSingle fiber was aligned either parallel (solid line) or perpendicular (dashed
13、 line) to the direction of polarization of laser beamRaman spectra of (A) major ampullate silk of Nephila edulis, and (B) degummed silk of Bombyx moriRaman spectra of (A) major ampullate silk of Nephila edulis, and (B) degummed silk of Bombyx moriAFM images of spider silk取向的微纤维结构取向的微纤维结构丝纤维长轴方向丝纤维长轴
14、方向TEM pictures of spider MA silkScale bar=0.2mThe micrographs of contracted spider silkCD experiment of silk fibroinThe regenerated silk fibroin solution was prepared by dissolving the degummed silk fiber into a 9.3 mol/L LiBr solution and then dialyzed against distilled water. Resultant regenerated
15、 silk fibroin concentration is about 1% (w/w), and then was diluted to 0.1% for CD experiment.The regenerated silk fibroin was demonstrated to have the same secondary structure (random coil) as that in the gland of silkwormJasco J-715 spectropolarimeter equipped with a slab (NESLAB RTE-111) Path len
16、gth of cell: 1mmTime range: 8 hrs190200210220230240250-12-10-8-6-4-202 Wavelength (nm)observed ellipticity obsTime resolved CD spectra of SF aqueous solution with and without -sheet “seeded”190200210220230240250-10-8-6-4-202468 observed ellipticity obsWavelength (nm) -sheet “seed” free19020021022023
17、0240250-10-8-6-4-202468observed ellipticity obsWavelenth (nm)190200210220230240250-10-8-6-4-202468 Wavelenth (nm)observed ellipticity obs5% -sheet “seed” added20% -sheet “seed” addedTime range: 0233 hoursConcentration of fibroin: 0.1% (wt/wt)Time and “seeded” dependence of conformation transition of
18、 fibroin190200210220230240250-8-6-4-20246Wave length (nm) 5 54 41. 0 hours1. 0 hours2. 6 hours2. 6 hours3. 18 hours3. 18 hours4. 74 hours4. 74 hours5. 233 hours5. 233 hoursA3 32 21 1 (10-3deg cm2/dmol)190200210220230240250-4-202461. 0 hours1. 0 hours2. 6 hours2. 6 hours3. 18 hours3. 18 hours4. 74 ho
19、urs4. 74 hours5. 233 hours5. 233 hours5544B332211 (10-3deg cm2/dmol)wavelength (nm)Original 0.1% silk fibroin solution20% -sheet seeded solution050100150200250-0.20.00.20.40.60.81.01.2ANormalized ellipticity 217Time course (hrs)The lag time in unseeded solutionSeed-free20% seed involved02468100.800.
20、850.900.951.001.051.10Normalized ellipticity 217time course (hrs)02550751000.00.20.40.60.81.0BNormalized ellipticity 217Time course (hrs)5% seed involved0.1 mg/ml silk fibroin solutionreveals a slower -sheet formation speed in a lower silk fibroin concentration with 20% -sheet seeded ( 0.01; 0.1 mg/
21、mL silk fibroin solution) Acoil A First-order dynamic process of aggregator growthLinear plots of ln(1-217) versus time course with different seed concentrations coilcoilAkdtAd05010015020025001234slope 0.0181 (0.0009) R 0.9748-ln(1- 217)Seed-free05010015020025001234slope 0.0179 (0.0010) R 0.9566-ln(
22、1- 217)5% seed05010015020025001234slope 0.0151 (0.0010) R 0.8941-ln(1- 217)time course (hours)20% seedln(1-nor217) = ktScheme of nucleation-dependent aggregation for silk fibroinWhy is the spin rate of nature silk so slow by comparison with synthetic fiber, but its molecular chain still has such hig
23、h orientation?shearing forcerandom coil unit -sheet unitnucleinucleation induced by silk pressaggregation growthTime-resolved FTIR spectra of silk fibroin membrane4000 3500 3000 2500 2000 1500 10000.00.51.01.52.0AbsorbanceFrequency (cm-1)Amide IAmide IIAmide A, BFREQUENCY (cm-1) ASSIGNMENT (Susi, 19
24、69)3300Amide A: NH stretch3100Amide B: NH stretch1690-1600Amide I: CO stretch1575-1480Amide II: NH stretch, CN stretch1301-1229Amide III: CN stretch, NH bendAmide I Secondary Structure Assignments:1620 - 1640 -sheet1644random coil (D2O)1648 - 1657-helix1665310 helix1670 - 1695 anti-parallel -sheet,
25、-turnSCAN PARAMETERS: regenerated silk fibroin membrane, 5m thick Nicolet Magna 860 FTIR spectrometer BaF2 liquid cell, path length 15 m 70% ethanol/30% D2O solution 64 scans per spectrum, 4 cm-1 resolution interval between two successive spectra: 5.85 sec MCT detectorAmide I band of regenerated Bom
26、byx mori silk fibroin(a) membrane without any treatment; (b) membrane immersed in 70% ethanol for 24h and then dried; (c) the first spectrum (at 0.1 min) in the dynamic measurement (curve Ablack: normal FTIR spectrum; curve Bblue: second derivative spectrum)17201700168016601640162016000.00.40.81.21.
27、6AbsorbanceWavenumbers (cm-1)0.00BA(b)Arbitrary Units 17201700168016601640162016000.00.51.01.52.0AbsorbanceWavenumbers (cm-1)0.00BA(a)Arbitrary Units 17201700168016601640162016000.00.40.81.21.62.0AbsorbanceWavenumbers (cm-1)-0.010.000.01BA(c)Arbitrary Units Time-resolved FTIR spectraFTIR spectra of
28、regenerated silk membrane during the conformation transition process from beginning to 60minDifference spectra obtained from left spectraConformation transition kinetics of B. mori fibroin membrane0102030405060-0.25-0.20-0.15-0.10-0.050.00(b)silk fibroin in 70% ethanol-helix at 1668 cm-1Data: Data2_
29、BModel: ExpDecay2Chi2 = 0.00001y0-0.231720.00086x000A10.148990.00592t10.518480.04195A20.109850.00561t25.261930.36352AbsorbanceTime (min)01020304050600.00.10.20.30.4(a)silk fibroin in 70% ethanol-sheet at 1618 cm-1Data: Data1_BModel: ExpDecay2Chi2 = 0.00003y00.327440.0011x000A1-0.27310.00919t10.52935
30、0.03448A2-0.103140.00942t24.509250.49955AbsorbanceTime (min)Fits with biphasic exponential decay functionsPossible three phases in the conformation transition of SF Notes: The time constants, , and the amplitudes were obtained by the absorbance difference spectral amplitudes with exponential decay f
31、unctions. The relative amplitudes 1 and 2 were calculated by 1=A1/(A1 + A2 ) and 2=A2/(A1 + A2 ), respectively (where A1 and A2 are the amplitudes of the first and the second phase.Conformation transition kinetics of B. mori silk fibroin membranes probe byabsorbance changes of -sheet band at 1618 cm
32、-1 and random coil band at 1668 cm-1IR marker (min)1(%) (min)2(%)-sheet band at 1618 cm-10.560.046745.200.47332Random coil at 1668 cm-10.510.056345.800.61373Burst phase: 50% of -sheet structure achieved Second process: 33% of -sheet formedThird process: “perfects” -sheet structure FTIR spectra of Ne
33、phila spidroin film during the conformation transition process Original FT-IR spectraThe conformation transition process induced by K+ from beginning to 240 min.Difference FT-IR spectraConformation transition kinetics of spidroin film probed by absorbance changes of -sheet band1620cm-1 1691cm-1 KCl
34、(mol/L) 1 (min) 2 (min) 1 (%) 1 (min) 2 (min) 1 (%) 1.0 7.42.4 53.39.5 517 7.82.1 54.96.2 437 0.5 4.52.5 49.88.2 439 4.02.3 53.92.2 337 0.3 3.70.2 48.67.0 315 3.00.2 57.75.9 216 (min) (min) 0.1 61.47.3 69.44.8 0.05 66.37.0 70.78.5 0.01 66.56.1 72.55.9 050100150200250-1.0-0.8-0.6-0.4-0.20.0(a) 1.0M 0
35、.5M 0.3M 0.1MAbsorbanceTime (min)050100150200250-1.0-0.8-0.6-0.4-0.20.0(b) 1.0M 0.5M 0.3M 0.1MAbsorbanceTime (min)蜘蛛丝优于蚕丝？！Materials Key repetitive sequences Fibroin Gly-Ala-Gly-Ala-Gly-Ser Spidroin (Ala)n, Gly-Gly-Gln, Gly-Ala-Gly, Gly-Gly-X 生物化学角度：蚕丝蛋白和蜘蛛丝蛋白的氨基酸组成和一级结构之间的差异很大原因？蜘蛛丝在一般情况具有高模、高强、高弹性
36、及很高的断裂吸收能等优异的综蜘蛛丝在一般情况具有高模、高强、高弹性及很高的断裂吸收能等优异的综合力学性能吸引科学家们试图大量地得到这一材料。合力学性能吸引科学家们试图大量地得到这一材料。Synthesis of spidroin？对于丝这一类非生理活性的蛋白质，一级结构是否能决定其最终性能对于丝这一类非生理活性的蛋白质，一级结构是否能决定其最终性能转基因山羊奶转基因山羊奶大肠杆菌或酵母大肠杆菌或酵母Nexia公司公司DuPont公司公司Biosilk类蜘蛛类蜘蛛丝蛋白丝蛋白(SGRGGLGGQGAGAAAAAAA)n及及(AAAGGAGQGGYGGLGSQGT)n蜘蛛丝蛋白的氨基酸基本组成序列
37、Stress-strain curves of recombinant silkNexia Biotechnologies Produced soluble recombinant (rc) dragline silk proteins with molecular masses of 60 to 140 kilodaltons by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. Spun monofilaments from a con
38、centrated aqueous solution of soluble rcspider silk protein under modest shear and coagulation conditions. The spun fibers exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. A. Lazaris etal, SCIENCE, 295, 2002 472-476Formation of regenerated
39、silksGuanidine-HClSelf-assembling of spidroinor dialysisNative SilkSolution8M12% aqueous solution Gel filtration / Protein aggregated on the surface of solution, the fiber-like regenerated silk could be drawn with the techniquesNuclear dependent aggregationRegenerated silk is also water insoluble.Mo
40、rphology of regenerated spider silkOriented fibrils or wrinklesCompared to the smooth surface of native silk drawn from the spider, the regenerated spider silk had a much more rougher surface. But notwithstanding the wrinkles along the long axis of the fiber, the surface of regenerated silk is still
41、 quite even and with an average diameter of about 9m only slightly thicker than that of native spider silk. Amino acid composition (mol%) of native spider silk proteins and regenerated silkThe amino acid compositions of our native and regenerated N. edulis silks were nearly identical. Guanidine hydr
42、ochloride only disrupts the non-covalent interaction in the proteins. Assumed that the primary structure of silk proteins experienced no serious changes during the dissolution of the native silk and its reformation. Thus the observed differences between both silks in mechanical properties would in m
43、ost probability be caused by their differences in secondary and/or condensed structure of the silk proteins.Amino acid Native N. edulis MA silk (reeled) Regenerated N. edulis silk N.clavipes spidroin (from MA gland)* N.clavipes MA silk (reeled)* ASP/ASN 1.5 0.7 1.9 1.06 THR 0.4 0.3 1.0 0.34 SER 1.7
44、1.8 3.0 2.24 GLU/GLN 12.8 13.2 10.1 11.02 PRO 9.4 6.7 1.7 2.04 GLY 38.2 39.9 40.3 49.96 ALA 24.0 25.7 28.4 22.71 CYC Not determined Not determined Not determined 0.06 VAL 0.7 0.6 1.5 0.89 MET 0.3 0.1 0.3 0.04 ILE 0.4 0.2 0.6 0.07 LEU 2.4 2.7 4.5 4.26 TYR 5.2 4.7 3.1 2.99 PHE 0.3 0.2 0.5 0.26 HIS Tra
45、ce 0.9 0.02 0.21 LYS 0.3 0.2 0.8 0.10 ARG 2.3 2.2 2.0 1.76 * Tillinghast, E. K. and Christenson, T. (1984) J. Arachnol., 12, 69-74 * Work, R. W. and Young, C. T. (1987), J. Arachnol., 15, 65-80 Stress-strain curves of regenerated silk0.000.050.100.150.200.250.300.350.400.000.020.040.060.080.100.120.
46、140.160.18streched in waterstretched in air Stress (GPa)Straininitial modulus : 6.0 GPa ;breaking strength: 0.11 to 0.14 Gpa; breaking elongations varied between 10% and 30%. neither the initial modulus nor the strength of our regenerated silk could compete with those of native dragline silk the rel
47、atively poor mechanical properties of our regenerated silk was due to its formation by a self-assembling process of molecular chain aggregation instead of the complex liquid crystal spinning of native silk Loading-unloading cycles of spider silks0.000.020.040.060.080.100.120.140.160.180.00.10.20.30.
48、40.50.60.70.8 regenerated silk native silk shrunk silkStress (GPa)Straina large degree of permanent setting after the first cycle showed in regenerated silksuggested that the molecular chains in the regenerated silk were rather disorientated Solid line: parallelDashed line: perpendicularRaman spectr
49、a of native and regenerated spider silkNative silkRegenerated silk:nearly isotropic -sheet and slightly difference between productsHierarchical structure of silks(Ala)n, n=47带丝胶的桑蚕丝蜘蛛丝从高分子凝聚态的角度看，蚕丝和蜘蛛丝的微观结构非常相似从高分子化学的角度看，蚕丝蛋白和蜘蛛蛋白的主链非常一致What can we do from point of view in polymer science动物丝素蛋白为结构性
50、纤维蛋白，其分子量约在300,000400,000之间，且没有明显的生理活性适合从高分子物理和化学的角度适合从高分子物理和化学的角度来进行理解和研究？！来进行理解和研究？！链结构，制备过程，聚集态结构，性能Dissected MA gland & associated structure (Nephila E.)Dissected silk gland of Bombxy moriSingle filament of spider silk in various conditionsStress-strain curves of Araneus d. dragline during (wet
