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.

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.

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.

Spiders have evolved proteins that can be kept in a highly concentrated soluble form in the silk gland yet rapidly assemble into stable silk fibers under certain environmental conditions. The transition between soluble and fibrillar states is partly regulated by the pH-sensitive N-terminal (NT) domain which has emerged as nature’s own solubility-enhancing domain. NT has an inherent capacity to keep the silk proteins’ partly hydrophobic and very aggregation-prone regions from premature fibrillation in spite of storage at enormous concentrations. The genetically engineered double-mutant NT* shows increased solubility and stability and has arisen as a powerful tool for the production of aggregation-prone as well as other recombinant proteins. Here we describe a robust and highly efficient protocol for improved soluble expression of peptides and proteins by fusion to the NT* tag.

Chronic kidney disease is associated with neurovascular complications including cognitive impairment, potentially involving blood–brain barrier (BBB) impairment. The protein-bound uremic toxin indoxyl sulfate (IS) promotes endothelial injury, but its effects on human brain microvascular endothelial cells remain incompletely understood. Human cerebral microvascular endothelial cells (hCMEC/D3) were exposed to IS 200 and 900 μM. BBB integrity was assessed by the FITC-dextran (4 kDa) transwell permeability assay and claudin-5 immunofluorescence. Transcriptional responses were quantified by qPCR for aryl hydrocarbon (AhR) target genes, oxidative stress-associated and inflammatory markers, senescence, and junction-associated genes. Senescence-associated phenotypic changes were evaluated by SA-β-galactosidase staining and cytokine array profiling. IS increased endothelial permeability at 24 and 48 h (~1.5-fold relative to control) without evidence of cytotoxicity and reduced claudin-5 staining intensity. IS strongly upregulated AhR target genes, including CYP1A1, CYP1B1, and CYP1A2. NFE2L2 and IDO1 increased, while NFKB1 remained unchanged. SA-β-gal positivity increased, accompanied by elevated GM-CSF and G-CSF secretion, while CDKN1A decreased at IS 900 µM and CDKN2A remained unchanged. CDH5 was downregulated, TJP1 increased at 900 µM, and CLDN5 remained unchanged. These findings indicate that IS exposure is associated with impaired BBB integrity, AhR-related transcriptional responses, oxidative stress-associated transcriptional changes, junctional remodeling, and senescence-like endothelial features. However, causal attribution to individual pathways requires inhibition or knockdown studies.