Weetman, S. L. | bioRxiv Science

bioRxiv2026

Structural evolution of a yeast amyloid in vivo is shaped by chaperones

Cryo-electron microscopy (cryo-EM) studies of amyloid fibrils have revealed endpoint structures of disease-relevant filaments and polymorphic intermediates formed during in vitro assembly of prion-like proteins. However, how transmissible prion or prion-like amyloids evolve during de novo formation and maturation in living cells remains unknown. Here, using the yeast prion [PSI+] as a model, we isolated Sup35NM amyloid fibrils from successive stages of [PSI+] maturation in Saccharomyces cerevisiae and characterised their near-atomic structures and population-level structural diversity by combining cryo-EM and atomic force microscopy. We show that intermediate and mature states differ in predominant fibril structure and the regions of the Sup35 sequence incorporated into the core, and that structural diversity decreases during maturation. Curing of [PSI+] at the mature state by guanidine hydrochloride (GdnHCl), which selectively inhibits ATPase activity of the chaperone Hsp104, restored both the predominant intermediate amyloid structure and the broader structural diversity characteristic of the intermediate state. In addition, Hsp104, Ssa1 (Hsp70) and Sis1 (Hsp40) associate differently with fibrils from the two states. Together, these findings provide direct structural evidence for amyloid evolution in vivo and support a chaperone-mediated mechanism of conformer selection within a polymorphic amyloid population.

molecular biology↗

bioRxiv2020

Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein

Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intend of engineering BMC-based metabolic modules for new functions in synthetic biology.

biophysics↗