The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.

During storage in the silk gland, the n-terminal domain (nt) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that nts from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer's disease. A designed variant of nt from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per liter bacterial culture than previously reported.Orb-weaving spiders manufacture up to seven different silks, e.g. dragline silk derived from major ampullate silk proteins (spidroins, MaSp) and flagelliform silk derived from flagelliform spidroins (FlSp). The various spidroins share a common architecture -a large core repetitive region capped by globular N-and C-terminal domains (NT and CT) 1 . The divergent and large aggregation-prone repetitive regions of the spidroins determine the mechanical properties of the respective spider silks, while the terminal domains regulate silk fiber formation 2,3 . Despite their high aggregation propensity the spidroins can be stored at extremely high concentrations (30-50% w/v) in the spider silk gland, solubilized by the NT domain 1,4 .The NT dimerizes upon a drop in pH, which is crucial for silk fiber formation 1,5 . To ensure solubility also at low pH and widen the applicability of NT as a solubility enhancing fusion partner, a charged-reversed mutant has been designed (referred to as NT* MaSp ) 6 . The previously reported NT* MaSp tag is derived from the NT domain of Euprosthenops australis MaSp1 and folds as a five-helix bundle 6,7 . NT* MaSp is a pH insensitive constitutive monomer, highly stable and extremely soluble, and has been successfully applied for efficient production and purification of, among others, lung surfactant protein analogs, cholecystokinin-58, human antimicrobial cathelicidin and a designed β-sheet protein 6,8 .Aggregation-prone proteins and peptides are associated with several neurodegenerative disorders, e.g. Alzheimer's disease (AD), the most prevalent form of dementia 9,10 . These proteins/peptides often exhibit high β-sheet propensity, which make them prone to aggregate and form insoluble amyloid fibrils 11 . These intrinsic properties of amyloid-forming proteins make high-yield biochemical production challenging, yet the availability of pure protein samples is crucial for studying protein self-assembly and its associated neurotoxicity in vitro and in vivo. This is probably one important reason behind the fact that, despite immense efforts, the exact mechanisms of Aβ self-assembly are still unknown 9-11 . Recent advances have howe...

SummaryThe interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.

Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide β17. The fusion protein NT*-β17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that β17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, β17 adopts a β-sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.