This article discusses the structure and function of important regulatory elements in spider silk, the presence of which is vital for thread formation. Using nuclear magnetic resonance spectroscopy (NMR), both the three-dimensional structure as well as important findings on the function of these protein domains could be determined for the first time.
To date, spider silk is one of the most tear-resistant biomaterials known, with properties that exceed modern high-performance synthetic materials by far. The evolutionary optimization of spider silk over more than 450 million years has created a material that is harder than steel, yet also elastic and flexible like rubber [1-3]. This variety is made possible by up to six different silk glands in the female spider, each of which produces different silk proteins with varying properties. The superb material properties, low weight and biocompatibility make spider silk a sought-after material.
This article primarily seeks to address the structure of N- and C-terminal domains in the very robust dragline thread of the spider. Based on this data, a thread formation model will finally be discussed, which reveals the essential function of both domains in the context of the overall silk proteins.
Properties of Different Silk Proteins
The spider uses silk threads from different silk glands for a variety of purposes [2,3]. For instance, the major ampullate (MA) silk gland produces silk for the robust dragline thread and the frame of the web (major ampullate spidroin (MAS)). An auxiliary thread (minor ampullate spidroin (MIS)) is produced by the minor ampullate (MI) gland, which is the first to be used, and which serves as a template for the final web. Apart from the frame, the spider web consists of flagelliform silk, which is produced in the flagelliform gland. This silk is very elastic, so that the kinetic energy of prey can be absorbed during capture. The spider also uses silk to wrap its eggs. This covering consists of an inner layer, which originates from the aciniform silk gland, and an outer layer, which is produced in the tubuliform gland.
Additionally, the spider requires a further material to stick together the individual threads and to coat the sticky spiral.
This material comes from the tubuliform gland. The spider then draws the required silk material from the respective gland, as required.
Silk proteins consist of long and repetitive amino acid sequence modules. A distinction is made between the modules that lend strength to the silk and those that lend elasticity . A hard silk thread contains a high percentage of crystalline areas, which non-covalently join together different protein chains, but which are non-tensile due to the rigid structure. The optimal amino acid sequences for generating these crystalline areas are polyalanine and polyalanine/glycine blocks which presumably form β-sheet-type structures. An elastic thread is formed when blocks, made, for example, from GPGQQ (G:Glycine, P:Proline, Q:Glutamine), are included in greater numbers [1-3]. In solution and probably also in the micelle storage form (see below), these repetitive structures remain in an unfolded state. There are, however, non-repetitive protein domains structured in solution at the N- and C-terminal ends of the silk proteins [2-5] (fig. 1). The structure and function of these areas of spider silk proteins were unknown until recently and are discussed below.
Structure and Function of the C-Terminal Non-Repetitive Domain
The region with the highest conserved amino acid sequence among the spider silk proteins is the C-terminus with at least 45 % identity [4,6] between the least related pairs in this group (fig. 2a). It was shown that the C-terminus is contained in the finished thread and is undifferentiated . On this basis, it has been assumed that this domain plays an essential role in controlling the solubility of silk proteins. This domain is also important for thread formation, which is induced by a change in the ionic composition and by mechanical stimuli . In the process, the repetitive sequence elements are aligned with one another, thereby enabling the formation of β-sheet structures. Despite a range of published work, the function of this domain remains unclear. Moreover, no three-dimensional structure has been achieved to date, due to the strong aggregation tendency and the low stability of these proteins.
Our method of choice is nuclear magnetic resonance spectroscopy (NMR), which allows the structure of molecules to be examined in solution (see below). As opposed to X-ray structural analysis, the proteins do not have to exist in the form of crystals, but can be investigated in their native-like environment.
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