Structure and dynamics of a folding intermediate of a spidroin C-terminal domain

• Applicants: Professorin Dr. Ute Hellmich; Privatdozent Dr. Hannes Neuweiler
• Subject Area: Biophysics ; Biochemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 540980616

Web spiders connect fibroins, so-called spidroins, into silk threads of extraordinary toughness. The sequences of spidroins evolved over hundreds of millions of years and display a unique architecture that gives rise to subdomains with distinct structural and functional features. A large central segment consists of repetitive peptide motifs and is capped by the highly conserved, globular N- and C-terminal domains (NTD and CTD) that form homo-dimers. The terminal domains provide solubility, connectivity, and induce phase and structural transitions through sophisticated mechanisms triggered by chemical stimuli in the animal’s spinning duct. Domain swapping is an important mechanism of protein oligomerization and a hallmark of spidroin CTD dimers that swap C-terminal helices. Very recently we found that domain swapping facilitates partial unfolding of CTDs in the distal, acidified zone of the spinning duct, and may thus play an important role in silk formation. However, the mechanism of unfolding and the structure of the partially folded CTD are elusive. There are striking similarities in mechanisms of protein misfolding in humans, leading to beta-sheet rich protein aggregates and neurodegenerative disease, and protein refolding of partially unfolded silk proteins, leading to beta-sheet rich fibres in spider silk synthesis, both involving domain-swapped, partially folded structures. In preliminary work we observed that the spidroin CTD from the black widow spider Latrodectus hesperus unfolds and forms a partially structured intermediate at pH 5, the pH present at the end of a spinning duct, through cold denaturation. Cold denaturation of proteins originates from the temperature-dependence of the hydrophobic effect and is rarely observable under physiological conditions at temperatures above freezing. Here, we will combine solution NMR spectroscopy with circular dichroism and photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) to investigate the structure and mechanism of formation of the folding intermediate of the spidroin CTD from L. hesperus. We will perform thermal and chemical denaturation experiments at various pH values, mimicking the pH changes within a spinning duct, as well as mutagenesis and sequence truncation studies. Our project will provide insights into fundamental pathways of folding and cold denaturation of a spider silk protein and shed new light onto the role of domain swapping in protein phase and structural transitions.

• DFG Programme: Research Grants