Novel Ramazzottius varieornatus small heat shock protein purification methods, their morphological diversity and chaperone analysis

Abstract

Ramazzottius varieornatus (R. varieornatus), a tardigrade species, possesses unique survival capabilities in extreme environmental conditions that are challenging for almost all other tardigrade species and living organisms. R. varieornatus survives through a reversible 'death-like' ametabolic state known as cryptobiosis in response to stress. While the most recent studies delve into the role of tardigrades' intrinsically disordered proteins (TDPs) in mitigating protein aggregation during cryptobiosis, there are no studies that have addressed the structure and function of the small heat shock proteins (sHSPs) in R. varieornatus in this context. The HSP20 chaperone protein family has properties of preventing protein aggregation and precipitation in response to stress such as heat. This gene family is distinguished by its high conservation throughout biological evolution, marked by the presence of the α-crystallin domain (ACD). Previous genomic and bioinformatic analyses, conducted both in the literature and by the Durham University bioinformatics lab, have identified the upregulation of novel small heat shock proteins (sHSPs) in R. varieornatus as a response to heat stress. Notably, three members of this family (HSP20-6, HSP20-5, and HSP20-3) were selected for the current study based on their most elevated expression levels in response to heat, as well as bioinformatics analysis and variations in their terminal domains.
The purification of the wild-type native structures of R. varieornatus HSP20-5 and HSP20-3 proved challenging using both conventional and modern methods for molecular chaperone purification. This study led to the development of a new purification protocol, namely the "Fast" purification method. This method proved to be successful in purifying the novel sHSPs, demonstrating improved separation profiles and enhanced protein yield. Additionally, the study revealed that the alkaline treatment purification method documented in the literature causes structural changes to HSP20-5 that compromise its oligomerisation function. In this study, transmission electron microscopy (TEM) and heat aggregation chaperone activity assays revealed that HSP20-6 exhibits chaperone characteristics most closely resembling those of CRYAB. However, there is a notable distinction of larger size and polydisperse nature of the HSP20-6 protein particles, ranging from 15 to 32 nm in diameter. Following purification, HSP20-5 manifests in both soluble (HSP20-5S) and insoluble forms (HSP20-5P). The soluble form, HSP20-5(S), displays protein particles with diameters ranging from 14 to 30 nm. Notably, when compared with CRYAB, this variant exhibits minimal chaperone activity among the newly characterised sHSPs. This study reveals the first naturally occurring filaments of the molecular chaperone of HSP20-3 that were not induced or treated chemically. The filaments can reach a length of 2540nm and have an average width of 12nm (sometimes bifurcating to 6nm). There were also protein particles with 14-26nm in diameter. HSP20-3 shows high chaperone activity in heat aggregation assay compared with CRYAB using alcohol dehydrogenase and malate dehydrogenase. The C-terminal domain of HSP20-3 plays a crucial role in both the chaperone function of HSP20-3 and the structural stability of the filament’s elongation.
Biological crowding triggers fibrous formation for HSP20-5 and a gel-like structure with HSP20-3 during purification, similar to that observed with some TDPs.