1、侈初A f環(huán)境友好高分子課程作業(yè)題目： vBiomimetic Fibers Made ofRecombinant Spidroins with the Same Toughness as Natural Spider Silk 論文翻譯化學(xué)與材料工程 學(xué) 院高分子專業(yè)學(xué)號(hào) XXXXXXX學(xué)生姓名KITTY& LC指導(dǎo)教師二 0 xx年 X 月vBiomimetic Fibers Made of Recombinant Spidroins with the Same Toughness as Natural Spider Silk 論文翻譯 環(huán)境友好高分子課程作業(yè) 文獻(xiàn)來源：ADVANCED

2、MATERIALS 卷：27 期：13 頁：2189-2194DOI: 10.1002/adma.201404234出版年：APR 1 2015iomimetic Fibers Made of Recombinant Spidroins with the Same Toughness as Natural Spider Silk 由重組蛛絲蛋白制得韌性與天然蛛絲一樣的仿生纖維Authors: Ani ela Heidebrecht; Lukas Eisoldt; Joha nnes Diehl; An dreas Schmidt; Martha Geffers; Gregor Lang; Th

3、omas ScheibelAbstractUsing a self-assembly of recomb inant spidro ins, biomimetic spinning dopes are produced and wet-sp un into fibers. Upon vary ing the molecular desig n of the un derly ing recomb inant spidr oins, the in flue nce of the amino- and carboxy-term inal doma ins, as well as the size

4、of the repetitive core doma in on fiber mecha ni cs, is determ in ed. Fiber tough ness upon biomimetic process ing equals and eve n slightly exceeds that of n atural on es.Keywords: biomimetic materials;phase separati on; spider silk;tough ness摘要本課題研究了利用重組蛛絲蛋白的自組裝，生產(chǎn)出仿生纖維紡絲原液并用濕法紡絲制得纖維的性能。根據(jù)改變底層重組蛛絲

5、蛋白的分子設(shè)計(jì)實(shí)驗(yàn)可知，氨基和羧基末端區(qū)域的影響，同重復(fù)的核區(qū)域大小對纖維力學(xué)性能的影響一樣，是起決定性作用的。基于仿生加工的纖維的韌性等同甚至略超過自然的纖維。關(guān)鍵詞：仿生材料；相分離；蜘蛛絲；韌性pider dragline silk exhibits extraordinary mechanical properties combining a moderate Sstrength with good extensibility resulting in a toughness exceeding that of all otherNatural or synthetic fibers.

6、 Although spider silk has been in the focus of research since decades, the mechanical properties, especially the toughness, of reconstituted man-made fibers have n ever reached those of n atural spider silk. The properties are based on the un derly ing spidersilk prote ins (spidr oin s), their self-

7、assembly and their explicit process ing. Here, two out of threepre-requisites for tough fibers are tackled; the con tributi on of in dividual spidr oin doma ins to assembly is an alyzed, and process ing of recomb inant spidro ins into fibers is show n. Fiber toughness upon processing equals and even

8、 slightly exceeds that of natural ones dependent on both the underlying proteins and preparation (biomimetic self-assembly) of the silk dope, although the overall stre ngth is lower based on the used and simplified sin gle-prote in set-up.蜘蛛的牽引絲擁有中等強(qiáng)度和良好的可延展性，表現(xiàn)出非凡的力學(xué)性能，導(dǎo)致其韌性超過其他所有的天然或合成纖維。蜘蛛絲幾十年以來一

9、直是研究的焦點(diǎn)，但人工再造纖維的力學(xué)性能尤其是韌性，從未達(dá)到天然蜘蛛絲的水平。這些性能是基于底層蛛絲蛋白(spidroi ns)的自組裝和顯式加工過程。對于堅(jiān)韌的纖維而言，三分之二的重要條件已經(jīng)被解決：分析出了蛛絲蛋白單體域組裝的貢獻(xiàn)，顯示出了重組蛛絲蛋白加工成纖維的過程。雖然基于使用和簡化單一蛋白質(zhì)結(jié)構(gòu)的組裝后其整體強(qiáng)度較低，但加工過的纖維其韌性等同甚至略超過基于底層蛋白及其制備的紡絲液(仿生自組裝)的天然纖維。Spider dragli ne silk has for long bee n in the focus of materials research mainly due to a

10、 toughno other fiber can accomplish. Spider major ampullate (MA) silk fibers, aka dragline silk, show a core-shell-structure, with the core comprising proteinaceous fibrils covered by a three-layered shell of minor ampullate (MI) silk, glycoproteins and lipids, with only a minor role for the mechani

11、cal properties of the fibers.1 The mechanics are mainly based on the protein fibrils comprising at least two proteins classified as MaSp1 and MaSp2 (MaSp, spidroin = spider fibroin), both of which are gen erally dist in guished by their proli ne content, which is sig ni fica ntly higher inMaSp2.2-4

12、MA spidroins have a molecular weight of 200 -350 kDa5, 6 and are composed of ahighly repetitive core domain flanked by amino- and carboxy-terminal domains with a distinet4 5sequenee. The core domain contains repeated (up to 100 times) amino acid modules of 40 -200 ami no acids3-5 composed of polyala

13、 nine stretches and glyci ne/proli ne-rich motifs. The stre ngth of n atural spider silk fibers is based on the polyala nine stretches stacked into-sheet7 resembli ng3nano crystallites which are embedded in an amorphous matrix, 1,8 based on the glyc in e/proli ne-rich9areas and being responsible for

14、 the fibers elasticity and flexibility. Thenumber of tandemly4arrayed glyci ne/proli ne-rich motifs (GPGXX, X = predo minan tly tyros in e,leuci ne, glutam ine)isdirectly conn ected to the exte nsibility of silks. MaSp2 has nine con secutive GPGXX-motifs in a single repeat unit, whereas flagelliform

15、 silk has at least 43 of these motifs in one unit 10 and is2the most extensible spider silk with 200% of elongation. Strikingly, the terminal domains are highly con served betwee n differe nt spider species and even betwee n silk types of in dividualspiders; they are composed of 100 -50 ami no acids

16、 and are folded into five-helix bun dles.11, 12The termi nal doma ins are assembly triggers en abli ng the spidroin storage at high concen tratio ns (up to 50% (w/v) in the ampulla of the spinning gla nd (resembli ng the so-called spinning dope) and play an important role during initiation of fiber

17、assembly. 13-16蜘蛛牽引絲因其有其他纖維不能達(dá)到的韌性長時(shí)間以來一直是材料研究的熱點(diǎn)。蜘蛛主要的MA絲纖維，即蜘蛛絲，顯示出了核殼結(jié)構(gòu)，核心由蛋白原纖構(gòu)成（包含MI絲三層殼結(jié)構(gòu)、糖蛋白和脂質(zhì)），對于纖維的力學(xué)性能只起到了很小的作用。纖維的力學(xué)性能主要是由兩種蛋白質(zhì) MaSp1和MaSp2 （ MaSp,spidroin即為蛛絲蛋白）構(gòu)成的蛋白原纖。這兩種 蛋白質(zhì)都含有脯氨酸，在MaSp2中的含量更高一些。2-4MA蛛絲蛋白的分子量可達(dá)200-350kDa5, 6，并且是由高度重復(fù)的核域和兩側(cè)以獨(dú)特的序列排列著的氨基、羧基的終端 域組成。核域包含重復(fù)的（大于100倍）4,5由40-

18、200個(gè)氨基酸（包括聚丙氨酸伸直鏈、甘氨酸/富脯氨酸結(jié)構(gòu)基元）3-5構(gòu)成氨基酸分子。天然蜘蛛絲纖維的強(qiáng)度是依賴于聚丙氨酸伸 直鏈堆疊成類似于嵌入無定形質(zhì)的納米微晶1, 8的3折疊，依賴于負(fù)責(zé)纖維的彈性和柔韌性的甘氨酸/富脯氨酸結(jié)構(gòu)基元9。串聯(lián)著排列的甘氨酸/富脯氨酸結(jié)構(gòu)基元（GPGXX , X主 要為酪氨酸、亮氨酸、谷氨酰胺）的數(shù)量，直接關(guān)系到絲的可延展性。MaSp2在一個(gè)簡單的重復(fù)單元里有連續(xù)的九個(gè)GPGXX結(jié)構(gòu)基元，而細(xì)長而柔軟的絲在一個(gè)單元里至少有43個(gè)這樣的結(jié)構(gòu)基元10，有著200%的伸長率，是最具延展性的蜘蛛絲2。值得注意的是，終端域在不同的蜘蛛之間具有高度的保守性，甚至在同一蜘蛛身

19、體內(nèi)不同類型的絲之間；他們由100-150個(gè)氨基酸組成，并折疊成五螺旋束11,12。終端域是使蛛絲蛋白以高濃度（重量 /體積超過50% ）存儲(chǔ)在紡絲腺的壺腹中（類似于所謂的紡絲液）的組裝觸發(fā)器，并且在纖 維開始組裝時(shí)發(fā)揮重要作的用13-16。It has been hypothesized that pre-assembly of spidroins in the gland is the cause for lyotropic liquid crystal behavior in vivo.17, 18 Upon passage of the spinning dope through th

20、e tapered S-shaped spinning duct, sodium and chloride ions are replaced by potassium and more kosmotropic phosphate ions (in duci ng salt in g-out of the spidr oin s).19, 20 In comb in ati on with shear-stress, emerg ing from pull ing the fibers from the spiders abdome n, the spidro ins assemble int

21、o a nematic phase,17 enabling formation and correct alignment of-sheet-rich structures1 Invitro, during storage of the spidroins at pH 8.0 micellar-like structures can be detected, strictly14depending on the presenee of the carboxy-terminal domainbased on the fact thatcarboxy-terminal domains form d

22、isulfide-linked parallel dimers,16 while the amino-terminal doma ins are mono meric at n eutral pH.21 Addi ng phosphate ions causes the non repetitive carboxy-term inaldoma into partially refold and subseque ntly expose hydrophobicareas,16necessary to initiate fiber assembly. Further, upon decreasin

23、g the pH to5.7, as foui the end of the spinning duct in vivo,22 dimerization of the amino-terminal domain in an22an tiparallel manner is triggered in vitro, yieldi ng head-to-tail dimers en abli ng the formatio n of an en dless n etwork connecting the nano crystall ine -sheet structures.4-16, 21假設(shè)，蛛

24、絲蛋白在腺內(nèi)發(fā)生預(yù)裝是因?yàn)橛袡C(jī)生物體內(nèi)的溶致液晶行為17,18。在紡絲液通過錐形的s型紡絲輸送管時(shí)，鈉離子和氯離子被鉀離子和磷酸鹽離子（誘導(dǎo)鹽析蛛絲蛋白）取代19,20。同時(shí)剪切應(yīng)力產(chǎn)生作用，自蜘蛛的腹部生產(chǎn)出絲，蛛絲蛋白開始組裝成液晶相17,使3富折疊結(jié)構(gòu)形成并恰當(dāng)排列16。在體外，蛛絲蛋白存儲(chǔ)在pH為值8.0的環(huán)境中時(shí)可以發(fā)現(xiàn)類膠束結(jié)構(gòu)，是因?yàn)轸然K端域的存在14，羧基終端域形成二硫化物連接形成了平行二聚體16而氨基終端域在中性環(huán)境中是以單體形式存在的21。那個(gè)添加磷酸離子使非重復(fù)性的羧基終端域的一部分再折疊，隨后裸露出疏水端16，對于引發(fā)纖維組裝很關(guān)鍵。進(jìn)一步，降低pH值約為5.7，就像

25、在有機(jī)生物體內(nèi)紡絲輸送管末端發(fā)現(xiàn)的一樣22;在體外氨基終端域里反平行二聚反應(yīng)被引發(fā)22，產(chǎn)生頭尾相連的二聚體形成無盡的網(wǎng)絡(luò)以連接類納米微晶的3折疊結(jié)構(gòu)14 -621。Even though plenty of artificial spider silk fibers have been produced in the past using different recomb inant or recon stituted spidr oins and spinnin g-tech niq ues, so far no fibers have bee n obta ined with mecha

26、 ni cal properties, i.e., tough ness, even getti ng close to that of n atural spider silk fibers.23盡管在過去已經(jīng)有人使用不同的重組或再造蛛絲蛋白及紡絲技術(shù)生產(chǎn)出了大量的人造蛛絲 纖維，但到目前為止，沒有一種人造蛛絲纖維獲得較好的力學(xué)性能，或是說韌性，更不要說接近天然蜘蛛絲纖維的性能了。23Here, we made use of previously established tech no logies to recomb inan tly produce spider silk-like pr

27、oteins based on the sequenee of garden spider (A.diadematus) MA spidroins. A. diadematus MA silk contains, in con trast to other in vestigated spider species, at least two MaSp2 proteins which are called A. diadematus fibroin 3 and 4 (ADF3 and ADF4). Here, based on consen sus seque nces of ADF3 (seq

28、ue nee accessi on nu mber: AAC47010) eight engin eered varia nts (eADF3) were desig ned for recomb inant product ion in E. coli, vary ing in len gth/nu mber of core repeats and presence/absenee of the amino- and/or carboxy-terminal domains (Figure 1A), in order to in vestigate the impact of in divid

29、ual doma ins on storage, spinning dope con diti ons, assembly, and mecha ni cal properties of fibers upon spinning.在這里，我們利用了前人建立的技術(shù)：重組產(chǎn)生基于已知花園蜘蛛(A. diadematus )的MA蛛絲蛋白序列的蛛絲蛋白。A. diadematus，與其他研究的蜘蛛物種做對比，包含至少兩種 MaSp2 蛋白質(zhì)，它們被稱為 A. diadematus fibroin 3 和 A. diadematus fibroin4 (ADF3 和 ADF4 )。在這里，基于ADF3

30、(序列檢索號(hào)：AAC47010 )的共有序列，設(shè)計(jì)出八個(gè)變體(eADF3) 在大腸桿菌體內(nèi)重組生成。這八個(gè)變體的不同之處在于：核重復(fù)單元的長度和數(shù)量、氨基酸 終端域與/或羧基終端域的有或無(圖 1A)，用以研究單元域存儲(chǔ)、紡絲液條件和紡絲纖維 的機(jī)械性能對紡絲的影響。A)NIL 10 m Tris/HCl, pH 8.0) solution prior to dialysis against PEG. Electrolytic conductivity measurements (Table S1, Supporting Information) showed no detectable am

31、ounts of salt after PEG-dialysis. Further, structural an alysis of recomb inant spider silk soluti ons before and after dialysis against PEG showed no change in protein structure2 Dialysis of a solution with low protein concentration against a phosphate-containing buffer induced a liquid -liquid pha

32、se separation of eADF3 variants comprising the carboxy-terminal domain NR3 into a low density phase and a “ se-concentrated high density micellar phas&14 yielding a dope named“ biomimetic from now The additi on of the phosphate ions in duces a partial refold ing of the carboxy-terminal domain, leadi

33、ng to initiation of protein assembly into micelles. Further, the presence of the carboxy-terminal domain is an important prerequisite for fiber self-assembly. Dynamic light scattering experiments on eADF3 high and low density phases revealed that the high den sity phase contains prote in oligomers (

34、MW 30 MDa), which were non covale ntly associated, whereas the low density phase only showed dimeric proteins. 26在以前，紡絲原液被認(rèn)為是紡絲的重要因素24。在自然界中，蛛絲的紡絲原液具有很高的濃度(重量/體積高達(dá)50%)。從技術(shù)上講，如此高的濃度可以通過分步濃縮溶液的方法 得以實(shí)現(xiàn)?！皞鹘y(tǒng)的”紡絲原液(CSD)是通過使用反聚乙二醇(PEG)滲析法簡單地去除 蛋白液中多余的水分，產(chǎn)出液濃度在10% (重量/體積)到17% (重量/體積)的紡絲原液。為了防止未指定的聚集現(xiàn)象出現(xiàn)，反聚乙

35、二醇(PEG)滲析之前先將100*10-3m氯化鈉添加到緩沖溶液(50X 10-3m三羥甲基氨基甲烷/鹽酸，pH值為8.0)中。電解電導(dǎo)率的測量(表 S1,輔助信息)表明在反 PEG滲析之后沒有檢測到大量的鹽。此外，重組蛛絲溶液的結(jié)構(gòu) 分析表明反PEG滲析前后其蛋白質(zhì)的結(jié)構(gòu)沒有發(fā)生變化25。用含磷酸鹽緩沖液的低蛋白質(zhì)濃度溶液的滲析，在低密度相和“自我濃縮”產(chǎn)生在此稱之“仿生”紡絲原液的高密度膠束 相中14，誘導(dǎo)含羧基端基結(jié)構(gòu)域 NR3的eADF3變異體的液-液相分離。磷酸根離子的添 加誘發(fā)羧基終端域里的一部分發(fā)生再折疊，導(dǎo)致蛋白質(zhì)開始組裝成膠束。此外，羧基終端域的存在對于纖維自組裝來說是一個(gè)重

36、要的先決條件。對eADF3的高密度相和低密度相進(jìn)行動(dòng)態(tài)光散射實(shí)驗(yàn)，實(shí)驗(yàn)表明eADF3的高密度相含有蛋白質(zhì)低聚物(分子量大于30 MDa ),通過非共價(jià)鍵相連接，而低密度相只含有二聚的蛋白質(zhì)。26While the phase-separatedbiomimetic spinning dopes (BSD) were stable foa y3s, the CSDgelled within a few hours due to nucleated fertilization of the proteins.27, 28相分離的“仿生”紡絲原液（BSD ）能夠穩(wěn)定地存在 3到5天，而傳統(tǒng)紡絲原液由

37、于蛋白質(zhì)的成核生長會(huì)在幾個(gè)小時(shí)內(nèi)會(huì)發(fā)生膠凝現(xiàn)象。27, 28Wet-sp inning of CSD (before gelati on started) by precipitati on of the spidr oins in a coagulati on bath containing a mixture of water and isopropanol, as used previously,29 typically yielded in homoge neous fibers (Figure S1, Support ing In formati on) and sometimes

38、short fiber fragme nts. The least homogeneous fibers were obtained from dopes comprising spidroins without the carboxy-terminal domain (AQ)i2, (AQ) 24, N1L(AQ) 12, N1L(AQ) 24). In contrast, spinning from BSD gen erally resulted in very homoge neous and long fibers. However, it was n ecessary to30imp

39、rove the performanee of the fibers by poststretching. Du et al. detected that the protein network structure (predominantly size and orientation of sheet crystals) changes substantially with the silk reeling speed and thus determines the mechanical properties of the silk fiber. In the n atural spinni

40、ng process the spider can con trol the reeli ng speed with its hind legs, poststretch ing the fiber as soon as it leaves the spinneret. While the formation of the liquid crystalline phase occurs quickly, the crystall ine phase is formed slowly, 31 and this phase tran siti on depe nds on the initial

41、concentration and supersaturation of the silk protein solution. Upon shearing as well as poststretching, the protein chains are extended and thus getting closer to each other. As the local protein concen tratio n is in creased, crystal nu cleati on betwee n the protein cha ins is triggered. A high r

42、eeling speed results in a high -sheet crystal nucleus density, leading to fibers containing smaller crystallites, but with an in creased crystal proporti on. 30 Concerning the orie ntati on of the 3-sheet crystals, it was observed that a high reeli ng speed in duces a better orie ntati on of the 3-s

43、heet crystals along the thread axis. Therefore, reeling and poststretching of the spiders silk fiber defines its mechanical properties. Similar observations were made with fibers from flexible polymers, where draw ing and postdraw stretch ing led to better mecha nical properties (e.g.,33higher ten a

44、city) due to better stra in alig nment.In our set-up, the recomb inant fibers werestretched up to 600% of their initial length directly after spinning to align the spidroins. The diameter of poststretched fibers spun from BSD was uniform throughout each in dividual fiber with a mea n diameter variat

45、i on and 100 xobject lenses and the software Leica V4.3. Birefringence was detected using polarizers at 0, 45 :and 90 and pictures were obtained with the camera Leica DMC2900. For scanning electron microscopy (SEM, Zeiss LEO 1530, 3 kV), fiber samples and fiber breaking edges were sputtered with a 2

46、 nm plati num coat ing (Cress ington 208HR high-resoluti on sputter coater) before imagi ng. Fourier tran sformatio n in frared (FTIR) spectroscopy measureme nts of the fibers were performed using a Bruker Tensor 27 (Bruker Optics, Ettlingen, Germany) with a Hyperion 1000 FTIR microscope. Alignment

47、of the sheet crystals was analyzed using a 15 x object lens with polarizing filters at 0 and 90 in transmission mode (IR polarizers supplied by Optometrics Corporati on). Spectra were plotted using Origin 8.1G (Origi nLab Corporati on, Northampt on, MA, USA).纖維分析：纖維用光學(xué)顯微鏡(Leica DMI3000B )分析，纖維顯示出缺陷能

48、被處理。纖維直徑是用20 x,40 x和100 x物鏡和軟件Leica V4.3確定。使用偏振器的雙折射在0,45,和90檢測和以照相機(jī)的Leica DMC2900獲得圖片。為掃描電子顯微鏡 (SEM , ZeissLEO 1530 , 3千伏)，在成像之前纖維樣品和纖維斷裂部用2納米鉑涂層(Cressington 208HR高分辨率濺射鍍膜機(jī))進(jìn)行鍍層。使用帶Hyperion 1000 FTIR顯微鏡的Bruker Tensor 27 (布魯克光譜，Ettlingen，德國)對纖維進(jìn)行傅立葉紅外光譜( FTIR)的測量。使用傳輸模式在 0和90的偏振濾光片 15X物鏡在(由Optometri

49、es Corporation提供的IR偏振片)的B片 晶體進(jìn)行取向分析。光譜用 Origin 8.1 (OriginLab公司，Northampton , MA，美國)繪制。Ten sile Test ing: For ten sile test ing fiber pieces were moun ted on to plastic sample holders with a 2 mm gap using superglue (UHU GmbH & Co. KG). Ten sile testi ng was performed using a BOSE-1Electroforce 3220

50、 with a 0.49 N load cell and a pulling rate of 0.04 mm s at 30% relative humidity. Mecha ni cal data were calculated con sideri ng real stress and real stra in data and using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA). For statistical analysis an unpaired two-sided t-test was per

51、formed for groups with similar variances and sample numbers were 10, except for poststretched N1L(AQ)2NR3 fibers spun from CSD (n = 7).拉伸試驗(yàn)：使用強(qiáng)力膠(UHU GmbH & Co. KG )對拉伸試驗(yàn)纖維片安裝到有2mm間隙塑料樣品架上。進(jìn)行拉伸試驗(yàn)時(shí)使用用一個(gè)0.49 N加載細(xì)胞和在30%的相對濕度拉伸速度 0.04mm s-1 的 BOSE Electroforce3220。使用 Microsoft Excel2010 ( Microsoft 公司，華

52、盛頓 州雷蒙德市，美國)分別計(jì)算出考慮真實(shí)應(yīng)力和真實(shí)應(yīng)變數(shù)據(jù)的機(jī)械數(shù)據(jù)。類似方差的和樣品數(shù)分別為10為統(tǒng)計(jì)分析用于除了用CSD (n =7)軸向拉伸N1L (AQ) 12NR3的不成對雙面t-試驗(yàn)中纖維紡絲。References參考文獻(xiàn)A.Sp onner, W. Vater, S. Mo najembashi, E. Un ger, F. Grosse,K. Weisshart,PLoS On e2007,2,e998.C. Y. Hayashi, N. H. Shipley, R. V . Lewis,Int. J. Biol. Macromol.1999,24, 271.a) P. A.

53、 Guerette, D. G. Gin zi nger, B. H. Weber, J. M. Gosli ne,Scie nce1996,272, 112; b)K. W. San ggaard, J. S. Bechsgaard,X. Fan g,J. Duan, T. F. Dyrlund, V. Gupta, X. Jia ng, L.Che ng, D. Fan, Y. Fen g,L. Han, Z. Hua ng, Z. Wu, L. Liao, V. Settepa ni, I. B. Th? gerse n,B. Van thournout, T. Wang, Y. Zhu

54、, P. Funch, J. J. Enghild,L. Schauser, S. U. Andersen, P.Villesen, M. H. Schierup, T. Bilde,J. Wan g,Nat. Com mun. 2014,5, 1.M. B. Hinman, R. V . Lewis,J. Biol. Chem. 1992,267, 19320.N. A. Ayoub, J. E. Garb, R. M. Tinghitella, M. A. Collin, C. Y. Hayashi,PLoS One2007,2,e514.a) G. C. Candelas, J. Cin

55、tron,J. Exp. Zool.1981,216,1; b) C. Jackson,J. P.O BrienMacromolecules1995,28,5975.B. L. Thiel, C. Vi ney,Scie nce1996,273,1480.a) J. Perez-Rigueiro, M. Elices, G. R. Plaza, G. V . Guinea,Macromolecules2007,40, 5360; b) A.Spo nner, E. Un ger, F. Grosse,K. Weisshart,Nat. Mater.2005,4, 772.a) A. E. Br

56、ooks, H. B. Stei nkraus, S. R. Nelso n, R. V . Lewis,Biomacromolecules2005,6,3095; b) Y. Liu, A. Sponner, D. Porter,F. Vollrath,Biomacromolecules2008,9, 116; c) K. Ohgo, T. KawaseJ Ashida, T. Asakura,Biomacromolecules2006, 7, 1210.C. Y. Hayashi, R. V. Lewis. Mol. Biol.1998,275, 773.a) R. J. Challis,

57、 S. L. Goodacre, G. M. Hewitt,Insect Mol.Biol.2006,15,45; b) M.Hedhammar, A. Rising, S. Grip,A. S. Martinez,K. Nordling, C. Casals, M. Stark, J. Joha nsso n,Biochemistry2008,47, 3407; c) A. Risi ng, G. Hjalm, W. En gstrom, J. Joha nsso n,Biomacromolecules 2006,7, 3120.D. Huemmerich, C. W. Helse n, S

58、. Quedzuweit, J. Oschma nn ,R. Rudolph, T. Scheibel,Biochemistry2004,43,13604.a) G. Askarieh, M. Hedhammar, K. Nordling, A. Saenz, C. Casals,A. Rising, J. Johansson, S.D. Knight,Nature2010,465, 236;b) L. Eisoldt,A. Smith, T. Scheibel,Mater.Today 2011,14, 80.L. Eisoldt, J. G. Hardy, M. Heim, T. R. Sc

59、heibel,J. Struct. Biol. 2010,170, 413.L. Eisoldt, C. Thamm, T. Scheibel,Biopolymers2012,97, 355.F. Hag n, L. Eisoldt, J. G. Hardy, C. Ven drely, M. Coles, T. Scheibel,H. Kessler,Nature2010,465, 239.a) D. P. Knight, F. Vollrath,Philos. Trans. R. Soc. London, Ser. B 2002,357, 219; b) F. N. Braun, C. V

60、iney,lnt. J. Biol. Macromol.2003,32,59.L. On sager,A nn. Ny. Acad. Sci.1949, 51,627.D. P. Knight, F. V ollrath,Naturwissenschaften2001,88,179.P. Papadopoulos,J. S?lter, F. Kremer,Eur. Phys. J. E: Soft MatterBiol. Phys. 2007,24, 193.a) F. Hagn, C. Thamm, T. Scheibel, H. Kessler,Angew.Chem. Int.Ed. 20