Preferential adsorption to air–water interfaces: a novel cryoprotective mechanism for LEA proteins

Abstract

Late embryogenesis abundant (LEA) proteins comprise a diverse family whose members play a key role in abiotic stress tolerance. As intrinsically disordered proteins, LEA proteins are highly hydrophilic and inherently stress tolerant. They have been shown to stabilise multiple client proteins under a variety of stresses, but current hypotheses do not fully explain how such broad range stabilisation is achieved. Here, using neutron reflection and surface tension experiments, we examine in detail the mechanism by which model LEA proteins, AavLEA1 and ERD10, protect the enzyme citrate synthase (CS) from aggregation during freeze–thaw. We find that a major contributing factor to CS aggregation is the formation of air bubbles during the freeze–thaw process. This greatly increases the air–water interfacial area, which is known to be detrimental to folded protein stability. Both model LEA proteins preferentially adsorb to this interface and compete with CS, thereby reducing surface-induced aggregation. This novel surface activity provides a general mechanism by which diverse members of the LEA protein family might function to provide aggregation protection that is not specific to the client protein.

Mechanisms of Desiccation Tolerance: Themes and Variations in Brine Shrimp, Roundworms, and Tardigrades

“…Amphipathic alpha helices are thought to stabilize molecular interactions by maintaining appropriate charge contact sites via opposite charged and non-polar faces of the helix (Giménez-Andrés et al, 2018). LEA proteins may also act as an interface between air and water to prevent protein aggregation (Yuen et al, 2019). The amphipathic structure of LEA proteins allows for diverse functions.…”

Structural properties and cellular expression of AfrLEA6, a group 6 late embryogenesis abundant protein from embryos of Artemia franciscana

“…While many questions still exist in terms of the varied functions of LEA proteins, in vitro and in vivo studies have contributed insights into their roles (for reviews, see Hand et al 2011;Hincha and Thalhammer 2012;Janis et al 2018a;Tunnacliffe and Wise 2007). For example, LEA proteins have been shown to protect lipid bilayers of various compositions during drying and freezing (Hundertmark et al 2011;Navarro-Retamal et al 2018;Steponkus et al 1998;Thalhammer et al 2014;Tolleter et al 2007Tolleter et al , 2010, preserve the activity of target enzymes (Boswell et al 2014a;Goyal et al 2005;Grelet et al 2005;Popova et al 2015), and prevent protein aggregation ("molecular shielding"; Boucher et al 2010;Goyal et al 2005;Yuen et al 2019). The gain of secondary structure (α-helix, β-sheet, turns) that accompanies the dehydration of some LEA proteins may allow them to perform separate/additional roles in the dried state (Goyal et al 2003).…”

New group of transmembrane proteins associated with desiccation tolerance in the anhydrobiotic midge Polypedilum vanderplanki

“…The location of genes of well-known effectors of anhydrobiosis within ARIds provides further evidence of this connection. LEA proteins of Group 3, whose genes are co-located with PvLil genes, are extensively studied in a wide variety of organisms, demonstrating both the association with water deficit and experimentally verified effects, such as reducing surface-induced aggregation of proteins, protection and stabilizing the lipid bilayers and mitochondria, increasing cytoplasmic conductivity of cells, reinforcing sugar glasses 9 , 25 – 28 . The latter include the transition from an intrinsically disordered to a structured state on dehydration, the reinforcement of trehalose-based glassy matrix and direct protection of proteins against desiccation-induced aggregation demonstrated for PvLEA22 protein 14 , 15 .…”

“…Damaging effects of desiccation in these animals are mitigated via interplay of numerous protective mechanisms, including the formation of biological glass (vitrification), “molecular shield” and anti-aggregation activity of some proteins and enhanced antioxidant activity 3 – 5 . Intrinsically disordered proteins (IDP’s) frequently participate at least in some of the protective mechanisms related to desiccation tolerance 5 – 9 . Anhydrobiotic animals share also such features as a small size which is typically less than 5 mm and an absence of internal skeletons, at least on anhydrobiotic life stages.…”