Lessons from nature — protein fibers

Silks are protein fibers with remarkable mechanical properties. The discovery of the structural features that govern these properties is a challenge for biochemistry and structural biology. This review summarizes the results of the biochemistry of silk proteins as well as the knowledge of the molecular biology of the respective genes. In addition, an overview is presented on the efforts to produce recombinant silk proteins by biotechnological techniques.     

Plasticity in major ampullate silk production in relation to spider phylogeny and ecology

Spider major ampullate silk is a high-performance biomaterial that has received much attention. However, most studies ignore plasticity in silk properties. A better understanding of silk plasticity could clarify the relative importance of chemical composition versus processing of silk dope for silk properties. It could also provide insight into how control of silk properties relates to spider ecology and silk uses. We compared silk plasticity (defined as variation in the properties of silk spun by a spider under different conditions) between three spider clades in relation to their anatomy and silk biochemistry. We found that silk plasticity exists in RTA clade and orbicularian spiders, two clades that differ in their silk biochemistry. Orbiculariae seem less dependent on external spinning conditions. They probably use a valve in their spinning duct to control friction forces and speed during spinning. Our results suggest that plasticity results from different processing of the silk dope in the spinning duct. Orbicularian spiders seem to display better control of silk properties, perhaps in relation to their more complex spinning duct valve.     

When galectins recognize glycans: from biochemistry to physiology and back again

In the past decade, increasing efforts have been devoted to the study of galectins, a family of evolutionarily conserved glycan-binding proteins with multifunctional properties. Galectins function, either intracellularly or extracellularly, as key biological mediators capable of monitoring changes occurring on the cell surface during fundamental biological processes such as cellular communication, inflammation, development, and differentiation. Their highly conserved structures, exquisite carbohydrate specificity, and ability to modulate a broad spectrum of biological processes have captivated a wide range of scientists from a wide spectrum of disciplines, including biochemistry, biophysics, cell biology, and physiology. However, in spite of enormous efforts to dissect the functions and properties of these glycan-binding proteins, limited information about how structural and biochemical aspects of these proteins can influence biological functions is available. In this review, we aim to integrate structural, biochemical, and functional aspects of this bewildering and ancient family of glycan-binding proteins and discuss their implications in physiologic and pathologic settings.     

高致病性猪繁殖与呼吸综合征病毒BJBLZ株序列剖析

预测与分析HP-PRRSV BJBLZ株序列特点,为进一步该毒株的研究做准备.利用分子生物学软件及服务器,对HP-PRRSV BJBLZ株全序列及各结构蛋白的生化特性、溶解性和核定位信号等进行了分析,结果显示HP-PRRSV BJBLZ株至少可划分12个非结构蛋白和6个结构蛋白;同源性比较显示GP3和GP5蛋白变异性高,M和N蛋白保守.GP3、GP5和M蛋白糖基化位点与参考株有变化,GP2、GP4和N蛋白糖基化位点与所有参考株一致.服务器预测显示,N蛋白的碱性氨基酸比例明显高于其他蛋白,总平均亲水值明显低于其他蛋白;GP2、GP3和GP4蛋白不稳定,GP5、M和N蛋白稳定.6个结构蛋白预测不溶性概率均占66％以上,GP3不溶性概率最高.可以利用以上一些不同于以往各代表株的特点,为PRRS的诊断和防治进行进一步的研究.     

Mapping domain structures in silks from insects and spiders related to protein assembly

The exceptional solubility in vivo (20-30%, w/v) of the silk proteins of insects and spiders is dictated by both the need to produce solid fibres with a high packing fraction and the high mesogen concentration required for lyotropic liquid crystalline spinning. A further design requirement for silk proteins is a strong predominance of hydrophobic amino acid residues to provide for the hydrophobic interactions, water exclusion, and beta-crystallite formation required to produce strong insoluble threads. Thus, the domain structure of silk proteins needs to enable nanoscale phase separation to achieve high solubility of hydrophobic proteins in aqueous solutions. Additionally, silk proteins need to avoid premature precipitation as beta-sheets during storage and processing. Here we use mapping of domain types, sizes and distributions in silks to identify consistent design features that have evolved to meet these requirements. We show that silk proteins consist of conspicuously hydrophilic terminal domains flanking a very long central portion constructed from hydrophobic blocks separated by hydrophilic ones, discussing the domain structure in detail. The general rules of construction for silk proteins based on our observations should give a useful guide to the way in which Nature has solved the problem of processing hydrophobic proteins in water and how this can be copied industrially. Following these rules may also help in obtaining adequate expression, soluble products and controllable conformational switches in the production of genetically engineered or chemically synthesized silk analogues. Thus these insights have implications for structural biology and relevance to fundamental and applied questions in material science and engineering.     

Chemical communication in proteobacteria: biochemical and structural studies of signal synthases and receptors required for intercellular signalling

Cell-cell communication via the production and detection of chemical signal molecules has been the focus of a great deal of research over the past decade. One class of chemical signals widely used by proteobacteria consists of N-acyl-homoserine lactones, which are synthesized by proteins related to LuxI of Vibrio fischeri and are detected by proteins related to the V. fischeri LuxR protein. A related marine bacterium, Vibrio harveyi, communicates using two chemical signals, one of which, autoinducer-2 (AI-2), is a furanone borate diester that is synthesized by the LuxS protein and detected by a periplasmic protein called LuxP. Evidence from a number of laboratories suggests that AI-2 may be used as a signal by diverse groups of bacteria, and might permit intergeneric signalling. These two families of signalling systems have been studied from the perspectives of physiology, ecology, biochemistry, and more recently, structural biology. Here, we review the biochemistry and structural biology of both acyl-homoserine-lactone-dependent and AI-2-dependent signalling systems.     

Single honeybee silk protein mimics properties of multi-protein silk

Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk.     

Novel assembly properties of recombinant spider dragline silk proteins

Spider dragline silk, which exhibits extraordinary strength and toughness, is primarily composed of two related proteins that largely consist of repetitive sequences. In most spiders, the repetitive region of one of these proteins is rich in prolines, which are not present in the repetitive region of the other. The absence of prolines in one component was previously speculated to be essential for the thread structure. Here, we analyzed dragline proteins of the garden spider Araneus diadematus, ADF-3 and ADF-4, which are both proline rich, by employing the baculovirus expression system. Whereas ADF-3 represented an intrinsically soluble protein, ADF-4 was insoluble in vitro and self-assembled into filaments in the cytosol of the host insect cells. These ADF-4 filaments displayed the exceptional chemical stability of authentic silk threads. We provide evidence that the observed properties of ADF-3 and ADF-4 strongly depend on intrinsic characteristics such as hydropathicity, which differs dramatically between the two proteins, as in most other pairs of dragline silk proteins from other Araneoidea species, but not on their proline content. Our findings shed new light on the structural components of spider dragline silk, allowing further elucidation of their assembly properties, which may open the door for commercial applications.     

Invited review the coiled coil silk of bees, ants, and hornets

In this article, we review current knowledge about the silk produced by the larvae of bees, ants, and hornets [Apoidea and Vespoidea: Hymenoptera]. Different species use the silk either alone or in composites for a variety of purposes including mechanical reinforcement, thermal regulation, or humidification. The characteristic molecular structure of this silk is α-helical proteins assembled into tetrameric coiled coils. Gene sequences from seven species are available, and each species possesses a copy of each of four related silk genes that encode proteins predicted to form coiled coils. The proteins are ordered at multiple length scales within the labial gland of the final larval instar before spinning. The insects control the morphology of the silk during spinning to produce either fibers or sheets. The silk proteins are small and non repetitive and have been produced artificially at high levels by fermentation in E. coli. The artificial silk proteins can be fabricated into materials with structural and mechanical properties similar to those of native silks.     

Controlling the Molecular Structure and Physical Properties of Artificial Honeybee Silk by Heating or by Immersion in Solvents

Honeybee larvae produce silken cocoons that provide mechanical stability to the hive. The silk proteins are small and non-repetitive and therefore can be produced at large scale by fermentation in E. coli. The recombinant proteins can be fabricated into a range of forms; however the resultant material is soluble in water and requires a post production stabilizing treatment. In this study, we describe the structural and mechanical properties of sponges fabricated from artificial honeybee silk proteins that have been stabilized in aqueous methanol baths or by dry heating. Aqueous methanol treatment induces formation of ß-sheets, with the amount of ß-sheet dictated by methanol concentration. Formation of ß-sheets renders sponges insoluble in water and generates a reversibly compressible material. Dry heat treatments at 190°C produce a water insoluble material, that is stiffer than the methanol treated equivalent but without significant secondary structural changes. Honeybee silk proteins are particularly high in Lys, Ser, Thr, Glu and Asp. The properties of the heat treated material are attributed to generation of lysinoalanine, amide (isopeptide) and/or ester covalent cross-links. The unique ability to stabilize material by controlling secondary structure rearrangement and covalent cross-linking allows us to design recombinant silk materials with a wide range of properties.     

The molecular aspects of absorption and metabolism of carotenoids and retinoids in vertebrates

Vitamin A is an essential nutrient necessary for numerous basic physiological functions, including reproduction and development, immune cell differentiation and communication, as well as the perception of light. To evade the dire consequences of vitamin A deficiency, vertebrates have evolved specialized metabolic pathways that enable the absorption, transport, and storage of vitamin A acquired from dietary sources as preformed retinoids or provitamin A carotenoids. This evolutionary advantage requires a complex interplay between numerous specialized retinoid-transport proteins, receptors, and enzymes. Recent advances in molecular and structural biology resulted in a rapid expansion of our understanding of these processes at the molecular level. This progress opened new avenues for the therapeutic manipulation of retinoid homeostasis. In this review, we summarize current research related to the biochemistry of carotenoid and retinoid-processing proteins with special emphasis on the structural aspects of their physiological actions. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

计算机模拟和X-射线实验研究蛋白质机械力学

近年来,研究发现在动力学反应及其模拟中,机械力主要表现为一个生理刺激元素。机械力如血管中的剪切流或肌肉组织中的伸缩力,能够紧密地控制着基因表达或组织修复等生物过程,在生物体中起着非常重要的作用。而对于这样的力控制生物过程的原因仍然未知。本文通过应用结构生物学、生物物理实验以及计算机模拟来展示如何研究蛋白质动力学。它主要综述了三个特定的主题：丝蛋白纤维动力学模拟、血管中蛋白质的剪切诱导过程以及在力的作用下酶的反应。     

Composition and hierarchical organisation of a spider silk

Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material.     

Molecules are more than markers: new directions in molecular microbial ecology

Macromolecules such as DNA, RNA, and proteins are widely used to quantify diversity in natural communities, to monitor the dispersal of organisms and their genes, and to trace phylogenetic relationships among organisms. With such widespread use of molecules as markers, it is easy to forget that they perform functions that are integral to the survival of organisms. The structural and functional properties of macromolecules have been intensively studied in genetics, biochemistry, and physiology. These fields, however, have not generally focused on the ecological significance of molecular variation, but instead have used defective variants as tools for identifying structure and function. Understanding the significance of molecular variation for organismal success or failure is a central problem in ecology, one whose solution will require the integration of diverse approaches and perspectives from ecology, molecular biology, genetics, physiology, and evolutionary biology. I review several studies of bacterial populations evolving in simple environments that have begun to integrate these approaches and perspectives in order to examine the consequences of molecular variation for ecological performance.     

Secondary structures and conformational changes in flagelliform, cylindrical, major, and minor ampullate silk proteins. Temperature and concentration effects

Orb weaver spiders use exceptionally complex spinning processes to transform soluble silk proteins into solid fibers with specific functions and mechanical properties. In this study, to understand the nature of this transformation we investigated the structural changes of the soluble silk proteins from the major ampullate gland (web radial threads and spider safety line); flagelliform gland (web sticky spiral threads); minor ampullate gland (web auxiliary spiral threads); and cylindrical gland (egg sac silk). Using circular dichroism, we elucidated (i) the different structures and folds for the various silk proteins; (ii) irreversible temperature-induced transitions of the various silk structures toward beta-sheet-rich final states; and (iii) the role of protein concentration in silk storage and transport. We discuss the implication of these results in the spinning process and a possible mechanism for temperature-induced beta-sheet formation.     

Structural conformation of spidroin in solution: a synchrotron radiation circular dichroism study

Spider silk is made and spun in a complex process that tightly controls the conversion from soluble protein to insoluble fiber. The mechanical properties of the silk fiber are modulated to suit the needs of the spider by various factors in the animal's spinning process. In the major ampullate (MA) gland, the silk proteins are secreted and stored in the lumen of the ampulla. A particular structural fold and functional activity is determined by the spidroins' amino acid sequences as well as the gland's environment. The transition from this liquid stage to the solid fiber is thought to involve the conversion of a predominantly unordered structure to a structure rich in beta-sheet as well as the extraction of water. Circular dichroism provides a quick and versatile method for examining the secondary structure of silk solutions and studying the effects of various conditions. Here we present the relatively novel technique of synchrotron radiation based circular dichroism as a tool for investigating biomolecular structures. Specifically we analyze, in a series of example studies on structural transitions induced in liquid silk, the type of information accessible from this technique and any artifacts that might arise in studying self-assembling systems.     

BAR, F-BAR (EFC) and ENTH/ANTH domains in the regulation of membrane-cytosol interfaces and membrane curvature

BAR and ENTH domains are families of alpha-helical lipid bilayer binding modules found in proteins that function in endocytosis, actin regulation and signaling. Several members of these families not only bind the bilayer, but also participate in the regulation of its curvature. These properties are thought to play physiological roles at sites of membrane budding and at other sites where narrow tubular membranes occur in vivo. Studies of BAR and ENTH domains and of their flanking regions have provided new insights into mechanisms of membrane deformation and curvature sensing, and have emphasized the importance of amphipathic helices, thought to intercalate in one of the leaflets of the lipid bilayer, in the generation of membrane curvature. Structural studies and database searches are rapidly expanding the BAR and ENTH domains families, with the identification of new related domains and subfamilies, such as F-BAR (also called EFC) domains and ANTH domains, respectively. Here we present a short overview of the properties of these domains based on evidence obtained from genetics, cell biology, biochemistry and structural biology.     

Sirtuins: multifaceted drug targets

Sirtuin (Sir2) proteins being key regulators of numerous cellular processes have been, over the recent past, the subject of intense study. Sirs have been implicated in diverse physiological processes ranging from aging and cancer to neurological dysfunctions. Studies on Sir2s using tools of genetics, molecular biology, biochemistry and structural biology have provided significant insight into the diverse functions of this class of deacetylases. This apart, medicinal chemistry approaches have enabled the discovery of modulators (both activators and inhibitors) of Sir2 activity of diverse chemical structures and properties. The availability of these small molecule modulators of Sir2 activity not only has pharmacological significance but also opens up the possibility of exploiting chemical genetic approaches in understanding the role of this multi-functional enzyme in cellular processes.     

Synthesis and characterization of multiblock copolymers based on spider dragline silk proteins

Spider dragline silk with its superlative tensile properties provides an ideal system to study the relationship between morphology and mechanical properties of a structural protein. Accordingly, we synthesized two hybrid multiblock copolymers by condensing poly(alanine) [(Ala)(5)] blocks of the structural proteins (spidroin MaSp1 and MaSp2) of spider dragline silk with different oligomers of isoprene (2200 and 5000 Da) having reactive end groups. The synthetic multiblock polymer displayed similar secondary structure to that of natural spidroin, the peptide segment forming a beta-sheet structure. These multiblock polymers showed a significant solubility in the component solvents. Moreover, the copolymer which contains the short polyisoprene segment would aggregate into a micellar-like structure, as observed by TEM.     

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。