In situ analysis of osmolyte mechanisms of proteome thermal stabilisation

ABSTRACT: Organisms across all kingdoms of life use small organic molecules called osmolytes to adapt to diverse environmental conditions and preserve viability. Osmolytes are thought to thermally stabilise proteins based on in vitro studies of a few purified proteins. The molecular mechanisms of osmolytes remain controversial and systematic global studies of osmolyte action within a complex cellular milieu are so far lacking. Here we present an in situ proteome-wide analysis of protein thermal stabilisation by osmolytes, covering all major classes of these molecules: trimethylamine N-oxide (TMAO), betaine, glycerol, proline, trehalose and glucose. Using a structural proteomics approach, we systematically probed the effects of osmolytes on protein thermal stability, native structures, and aggregation, thus revealing widespread mechanisms of osmolyte action, but also osmolyte- and protein- specific effects. All tested osmolytes stabilised the majority of the proteome. The stabilisation of most proteins could be explained by the preferential exclusion model, according to which osmolytes unfavourably interact with protein backbones thus promoting folded states. We also detected specific protein-osmolyte binding events which resulted in strong thermal stabilisation, although binding was not necessary for stabilisation. Aggregation of most proteins occurred at higher temperatures in the presence of osmolytes than in their absence. TMAO however promoted aggregation of small, unfolded proteins but also showed the strongest protein thermal stabilisation effects, which might reflect adaptation to the extreme living conditions of marine organisms that accumulate TMAO. Our global in situ analysis provides mechanistic insight into how osmolytes function within a complex biological matrix. Our data further provides a resource to guide the design or selection of stabilisers for protein-based drugs and the identification of conditions that increase thermotolerance of microorganisms in biotechnological applications.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion Lumos

ORGANISM(S): Escherichia Coli

SUBMITTER: Monika Pepelnjak  

LAB HEAD: Paola Picotti

PROVIDER: PXD036186 | Pride | 2024-08-09

REPOSITORIES: pride