原子力显微镜下丝素纤维及丝素蛋白的形态结构研究

利用原子力显微镜(AFM)、透射电镜(TEM)、圆二色谱仪(CD)等手段,研究了家蚕丝素纤维及丝素蛋白的形态结构,并尝试通过改变丝素蛋白溶液的酸碱性来观测其形态变化。结果表明,丝素纤维表面有许多沟槽和条纹,具有原纤结构特征;许多直径为20～50nm的圆形或椭圆形颗粒分子形成丝素蛋白的微观形态。在不同的酸碱条件下,球状颗粒分子具有不同的聚集方式,形成不同的微观形态。     

介导新生肽链折叠的胞质分子伴侣结构、功能及作用机制研究进展

分子伴侣是一类能够识别非天然蛋白并能协助其正确折叠、组装和转运的功能蛋白。最新研究发现,在原核或真核细胞中,不同结构、不同种类的分子伴侣形成了一个复杂的折叠系统,通过这个系统,蛋白质完成了从初步合成到形成具有生物活性的三维构象的过程,避免了折叠过程中多肽链的错误折叠、蛋白沉淀和有害物质的产生。文章综述了蛋白质折叠过程中不同种类分子伴侣组件的结构、功能和作用机制的研究进展,这些分子伴侣包括Hsp70、核糖体结合因子、伴侣素、前折叠素与Hsp90,并阐述了它们在蛋白质内稳态中的作用。     

对渗透现象的一种解释

提出溶液中溶质分子的热运动以热弹性振动为主,只有极少一部分高能分子才可以定向移动的假说。溶质分子的热振动会产生压力,而溶液压力是其中热振动的溶质分子产生的压力与连通的溶剂压力的叠加作用,因而溶液中的溶剂的压力总是比溶液的宏观压力低;渗透现象以及溶液的蒸气压降低、冰点下降现象正是由于溶液中溶剂的压力比溶液本身的宏观压力低而造成的。     

二级结构形成：蛋白质折叠起始过程的框架模型

框架模型认为二级结构形成是蛋白质起始过程的结构基础.文章介绍蛋白质同源片段的溶液构象及其构象研究法和多肽二级结构的从头设计,并综述这些研究成果应用于折叠起始过程的理论模型和蛋白质折叠起始过程的最新研究进展.     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

蜘蛛丝蛋白研究进展

由于蜘蛛丝蛋白分子高度重复的一级结构、特殊的溶解特性和分子折叠行为以及具有形成非凡力学特性丝纤维的能力而引人注目。本文从蛛丝蛋白基因、天然蛛丝形成过程、蛛丝蛋白的基因工程生产及蛛丝蛋白的应用前景等几个方面着重介绍了近20年来对蛛丝蛋白的研究进展。围绕蛛丝蛋白展开的研究将有助于揭示蛋白质一级结构、蛋白质分子折叠与蛋白质大分子特性之间的内在联系。     

不同因素对人Ⅹ型磷脂酶A2 包涵体重折叠的影响

人 Ⅹ型磷脂酶 A2 在大肠杆菌中的表达产物完全以不溶的包涵体形式存在 . 用以前适用于人胰型 (IB 型 ) 磷脂酶 A2 的稀释重折叠方法,并不能使它有效重折叠 . 以初步纯化的包涵体为对象,发现溶液的温度、 pH 值和蛋白质浓度对 Ⅹ 型磷脂酶 A2 体外重折叠有很大的影响 . 研究了一些小分子化合物对 Ⅹ 型磷脂酶 A2 ,在高蛋白质浓度 (1 g/L) 下重折叠的影响,发现 1 mol/L 的 L- 精氨酸能提高其重折叠效率达 6 倍多 . L- 精氨酸的结构类似物 L- 瓜氨酸对 Ⅹ 型磷脂酶 A2 的重折叠有较弱的改善,而 L- 赖氨酸和 L- 精氨酸甲基酯则降低了 Ⅹ 型磷脂酶 A2 的活性恢复 . 结果表明 , L- 精氨酸对蛋白质重折叠的帮助作用,可能是通过精氨酸与折叠中间物的结合产生的,精氨酸的胍基和羧基都是必需的,其侧链胍基以其特殊的带电方式阻遏了重折叠过程中的积聚和分子间二硫键形成的反应 .     

不同因素对人Ⅹ型磷脂酶A2包涵体重折叠的影响

人Ⅹ型磷脂酶A2 在大肠杆菌中的表达产物完全以不溶的包涵体形式存在. 用以前适用于人胰型(IB 型) 磷脂酶A2 的稀释重折叠方法,并不能使它有效重折叠. 以初步纯化的包涵体为对象,发现溶液的温度、pH 值和蛋白质浓度对Ⅹ型磷脂酶A2 体外重折叠有很大的影响. 研究了一些小分子化合物对Ⅹ型磷脂酶A2 ,在高蛋白质浓度(1 g/L)下重折叠的影响,发现1 mol/L 的L-精氨酸能提高其重折叠效率达6倍多. L-精氨酸的结构类似物L-瓜氨酸对Ⅹ型磷脂酶A2 的重折叠有较弱的改善,而L-赖氨酸和L-精氨酸甲基酯则降低了Ⅹ型磷脂酶A2 的活性恢复. 结果表明,L-精氨酸对蛋白质重折叠的帮助作用,可能是通过精氨酸与折叠中间物的结合产生的,精氨酸的胍基和羧基都是必需的,其侧链胍基以其特殊的带电方式阻遏了重折叠过程中的积聚和分子间二硫键形成的反应.     

纳米丝素蛋白微球的制备及性能研究

以家蚕丝素蛋白为原料,基于丝素自组装理论,通过酶解-干燥-溶解法制备不同尺寸的丝素蛋白微球,制备出的微球具有良好的水不溶性和稳定的分散性。对微球的形貌和结构表征结果表明,用该方法制备的丝素蛋白微球为纳米微球,当酶的添加量为2%且蛋白自组装时间为4 h时,丝素蛋白微球的平均粒径最小,仅为(32±11)nm。红外光谱(FT-IR)和X射线衍射(XRD)结果显示,微球中β-折叠结构的多少决定了微球晶体的大小,β-折叠越多,微球中晶体的体积越大。通过调控丝素蛋白自组装过程,可以制备平均粒径在30～140 nm之间的纳米丝素蛋白微球,且不引入任何有机溶剂和无机溶剂,制备过程绿色环保,制备出的丝素蛋白微球安全无毒。     

分子伴侣的多重功能

分子伴侣(molecular chaperone)在原核生物和真核生物的细胞中广泛存在.分子伴侣可稳定未折叠或部分折叠的多肽,并防止不适当的多肽链内或链间相互作用;有些分子伴侣也可与天然构象的蛋白质相互作用以促使寡聚态蛋白质发生结构重排.基于分子伴侣能识别并调节细胞内多肽的折叠,因此它们还具有介导线粒体蛋白跨膜转运,调控信息传导通路和转录、复制,以及参与微管形成与修复等功能.     

New process to form a silk fibroin porous 3-D structure

A new process to form fibroin spongy porous 3-D structure is reported herein. The process involves freezing and thawing fibroin aqueous solution in the presence of a small amount of an organic solvent. The process requires no freeze-drying, chemical cross-linking, or the aid of other polymeric materials. The solvent concentration, fibroin concentration, freezing temperature, and freezing duration affect the sponge formation, its porous structure, and its mechanical properties. Measurements by XRD and FTIR indicate that silk I and silk II crystalline structures exist in the fibroin sponge and that the secondary structure of fibroin is transformed to a beta-sheet from a random coil during this process. The tensile strength decreased slightly, but the fibroin sponge showed no deformation after autoclaving. Therefore, the fibroin sponge was sterilized using an autoclave. For 3 weeks, MC3T3 cells proliferated in the sterilized fibroin sponge. The fibroin sponge formed by this new process is applicable as a tissue-engineering scaffold because it is formed from biocompatible pure silk fibroin and offers both porous structure and mechanical properties that are suitable for cell growth and handling.     

Modelling self assembly of natural silk solutions

We present a tentative interpretation of the origin of nematic liquid crystalline order exhibited by various natural silk fibroin solutions, notably those of orb-weaving spiders and the domestic silkworm Bombyx mori. It is thought that liquid crystalline rheology is exploited during the spinning process. We discuss in this approach the response of the liquid crystalline phase diagram to equilibrium physiological conditions and to parameters characterising the amino acid sequence of the fibroin molecules. The phase diagram is sensitive in this latter respect to sequence mutations, such that it may constitute a source of evolutionary selection pressure.     

Bombyx mori silk fibroin liquid crystallinity and crystallization at aqueous fibroin-organic solvent interfaces.

A banded morphology has been observed for Bombyx mori silk fibroin films obtained from an aqueous hexane interface; the period of the banding is approximately 1 microm. Morphology and diffraction from different regions of the banded structure suggest that it is a free surface formed by a cholesteric liquid crystal. Truncated hexagonal lamellar crystallites of B. mori silk fibroin have been observed in films formed in the surface excess layer of fibroin at the interface between aqueous fibroin and hexane or chloroform. Based on initial crystallographic evidence, a three-fold helical conformation has been ascribed to the fibroin chains within the crystals. The chain conformation and crystalline habit appear to be similar to the silk III structure previously observed at the air-water interface (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Valluzzi R, Gido S, Zhang W, Muller W, Kaplan D. Macromolecules 1996;29:8606-8614) but the crystalline packing is different. Diffraction data obtained for the crystallites are similar to diffraction behavior for a collagen-like model peptide. Diffraction patterns obtained from crystallized regions of the banded morphology can be indexed using the same unit cell as the hexagonal lamellar crystallites. Surfactancy of fibroin and subsequent aggregation and mesophase formation may help to explain the liquid crystallinity reported for silk, which is long suspected to play a role in the biological silk spinning process (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Willcox, P. J.; Gido, SP, Muller W, Kaplan DL. Macromolecules 1996:29:5106-5110; Magoshi J, Magoshi Y, Nakamura S. In: Kaplan D, Adams W, Farmer B, Viney C, editors, Mechanism of Fiber Formation of Silkworm. Washington, DC: American Chemical Society 1994:292-310; Magoshi J, Magoshi Y, Nakamura S. J Appl Polym Sci Appl Polym Symp 1985;41:187-204; Magoshi J, Magoshi Y, Nakamura S. Polym Commun 1985;26:309.).     

A study on the flow stability of regenerated silk fibroin aqueous solution

The flow stability of silk fibroin (SF) aqueous solutions with different concentrations under different temperatures was investigated. It was found that the flow stability decreased quickly with the increase of solution concentration and temperature. X-ray diffraction, Fourier transform infrared (FTIR) and Raman spectroscopy analysis showed that silk fibroin in aqueous solution was mainly in random coil and alpha-helix conformation. However, it turned into alpha-helix and beta-sheet conformation after gelation, and both silk I and silk II crystalline structures appeared accordingly. The investigation implies that the original dilute regenerated SF aqueous solution should be stored under low temperature and concentrated just before spinning.     

Swelling and dissolution of silk fibroin (Bombyx mori) in N-methyl morpholine N-oxide.

Bombyx mori silk fibers were dissolved in N-methyl morpholine N-oxide (MMNO), an organic cyclic amine oxide used for the solvent spinning of regenerated cellulosic fibers. The commercial MMNO monohydrate used in this study as a solvent for silk is a hygroscopic compound crystalline at room temperature, which becomes an active solvent after melting at 76 degrees C. The degree of hydration of MMNO was checked by DSC measurements. The solvation power of MMNO towards silk fibroin drastically decreased at a water content > or = 20-21% w/w. Dissolution of silk required both thermal and mechanical energy. The optimum temperature was 100 degrees C. At lower temperatures dissolution proceeded very slowly. At higher temperatures, rapid depolymerization of silk fibroin occurred. The value of the Flory-Huggins interaction parameter chi for the MMNO-H2O-silk fibroin system was -8.5, suggesting that dissolution is a thermodynamically favored process. The extent of degradation of silk fibroin was assessed by measuring the intrinsic viscosity and determining the amino acid composition of silk after regeneration with an aqueous methanol solution, which was effective in removing the solvent and coagulating silk. Regenerated silk fibroin membranes were characterized by infrared spectroscopy, differential scanning calorimetry and scanning electron microscopy. The prevailing molecular conformation of silk fibroin chains was the beta-sheet structure, as shown by the intense amide I-III bands at 1704, 1627, 1515, 1260, and 1230 cm(-1). The value of the I1260/I1230 intensity ratio (crystallinity index) was 0.68, comparable to that of the fibers. The DSC thermogram was characteristic of a silk fibroin material with unoriented beta-sheet crystalline structure, with an intense decomposition endotherm at 294 degrees C. The SEM examination of fractured surfaces showed the presence of a dense microstructure with a very fine texture formed by densely packed roundish particles of about 100-200 nm diameter.     

In vitro study of the proteolytic degradation of Antheraea pernyi silk fibroin

In this study, Antheraea pernyi silk fibroin (Ap-SF) films were incubated with Protease Type XXI from Streptomyces griseus, at 37 degrees C, to investigate the degradation behavior in an in vitro model system. The enzyme-resistant fractions of Ap-SF films and the soluble peptides formed by proteolytic degradation were collected at specified times, from 1 to 17 days, and analyzed by high performance liquid chromatography, differential scanning calorimetry, FT-Raman, and FT-IR spectroscopy. Proteolysis resulted in extensive weight loss and progressive fragmentation of films, especially at long degradation times. A range of soluble peptides was formed by proteolysis. By high performance-size exclusion chromatography it was found that their average molecular weight changed with the time of incubation. The chemical analysis of the enzyme-resistant fraction of Ap-SF films at different times of degradation indicated that the proteolytic attack preferentially occurred in the less ordered Gly rich sequences and that the contribution of the Ala rich crystalline regions to the composition of biodegraded films became progressively larger. Accordingly, DSC and spectroscopic results showed an enhancement of the crystalline character of the biodegraded films. From the behavior of the most important thermal transitions, it was deduced that the alpha-helix domains probably represent the most enzyme-resistant fraction. The in vitro approach used in the present study seems to be a valid tool for studying the rate and mechanism of degradation of Ap-SF films and of other biopolymers of potential biomedical utility.     

Conformational transition and liquid crystalline state of regenerated silk fibroin in water

The conformational transition of molecular chains of regenerated silk fibroin (SF) aqueous solution is systematically investigated by circular dichroism, Raman, IR, and UV-vis spectroscopies. It is found that an initial random coil conformation of the SF can be readily changed into an ordered beta-sheet structure by optimizing the solution conditions, such as the SF concentration, pH, temperature, or metal-ion content. Circular dichroic spectra quantitatively confirm a steadily decreased content of the random coil conformation but a significantly increased beta-sheet content after an ultrasonic or extruding treatment. Furthermore, the extrusion is more powerful to achieve high beta-sheet content than the ultrasonic. It is interesting that the polarized optical micrographs of the SF aqueous solution extruded by injection illustrate the formation and existence of liquid crystalline state. A study of extrusion in vitro could be used as a model system to understand the natural silk spinning process in silkworm.     

Structural study of irregular amino acid sequences in the heavy chain of Bombyx mori silk fibroin

Recently, genetic studies have revealed the entire amino acid sequence of Bombyx mori silk fibroin. It is known from X-ray diffraction studies that the beta-sheet crystalline structure (silk II) of fibroin is composed of hexaamino acid sequences of GAGAGS. However, in the heavy chain of B. mori silk fibroin, there are also present 11 irregular sequences, with about 31 amino acid residues (irregular GT approximately GT sequences). The structure and role of these irregular sequences have remained unknown. One of the most frequently appearing irregular sequences was synthesized and its 3-D solution structure was studied by high-resolution 2-D NMR techniques. The 3-D structure determined for this peptide shows that it makes a loop structure (distorted omega shape), which implies that the preceding backbone direction is changed by 180 degrees, i.e., reversed, by this sequence. This may facilitate the beta-sheet formation between the crystal-forming building blocks, GAGAGS/GY approximately GY sequences, in the fibroin heavy chain.     

Diepoxide-triggered conformational transition of silk fibroin: formation of hydrogels

Silk fibroin hydrogels with tunable properties could be obtained from aqueous fibroin solutions (4.2 w/v %) in a short period of time. This was achieved by the addition of ethylene glycol diglycidyl ether (EGDE) into the reaction solution. Introduction of EGDE cross-links between the fibroin molecules decreases the mobility of the chains, which triggers the conformational transition from random-coil to β-sheet structure and hence fibroin gelation. Dynamic rheological measurements conducted at 50 °C show the formation of strong to weak hydrogels depending on the pH of the reaction solution. Although EGDE attacks the amino groups of fibroin and forms interstrand cross-links, β-sheets acting as physical cross-links dominate the elasticity of the hydrogels. Mechanical response of low-modulus fibroin hydrogels formed above pH 9.7 is highly nonlinear with strong strain hardening behavior (700%) arising from the alignment of the crystallizable amino acid segments.     

丝素蛋白/羟基磷灰石复合物对磷酸钙骨水泥性能的影响

目的：磷酸钙骨水泥（Calcium phosphate cement,CPC）以其诸多优点正得到了越来越多的应用,但其较差的力学性能表现也限制了它的使用范围。本研究目的在于改善磷酸钙骨水泥的力学性能,同时评估改性后的磷酸钙骨水泥的其他性能。方法：通过丝素蛋白（Silk fibroin,SF）的矿化自组装方法制备丝素蛋白／羟基磷灰石复合物（silk fibroin/hydroxyapitite composite, SF/HA）。按照1％、2％、3％、4％的质量分数加入磷酸钙骨水泥中,与磷酸钙骨水泥组对比。比较内容包括力学强度、抗渍散性能及细胞毒性。结果：以丝素蛋白溶液为液相组的磷酸钙骨水泥强度大约为35MPa。随后随着添加丝素蛋白／羟基磷灰石复合物的质量分数从1％增至3％,磷酸钙骨水泥的强度逐渐增加（P〈0．05）,最高约至45MPa。而当丝素蛋白／羟基磷灰石的质量分数达到4％时,磷酸钙骨水泥的强度较质量分数3％组小幅度下降至43MPa（P〈0．05）。以丝素蛋白溶液作为液相时,磷酸钙骨水泥的抗溃散能力也得到了加强。在MTT法测定细胞活力的对照实验中,无论是加入丝素蛋白溶液或丝素蛋白／羟基磷灰石复合物,都未观察到细胞毒性。结论：在磷酸钙骨水泥中加入3％质量分数的丝素蛋白／羟基磷灰石复合物,能显著提高磷酸钙骨水泥的抗压强度。而丝素蛋白溶液作为液相可改善磷酸钙骨水泥的抗溃散能力。同时,丝素蛋白和丝素蛋白／羟基磷灰石复合物都不表现出细胞毒性。更理想的力学强度和更强的抗溃散能力,大大扩展了磷酸钙骨水泥的应用范围。