Stability Comparisons between Natural versus Engineered Archaeal Heat-Shock Proteins

Abstract

AbstractCrenarchaeal group II chaperonins (a.k.a., “heat shock” proteins, HSPs) are abundantly expressed in species of the family Sulfolobaceae. HSPα and HSPβ expression is upregulated during thermal shock. HSPs are subunits of larger octadecameric complexes that function to protect intracellular proteins during thermal stress. Engineered HSPs have been constructed with the idea of protecting enzymes in industrial reactions. HSPβ-coh, a fusion protein comprised of HSPβ and type 1 cohesin from Clostridium thermocellum was used for proof-of-concept. Dockerin-endowed cellulolytic enzymes bind to the complex via cohesin-dockerin interactions. Enzymatic activity (i.e., hydrolysis of lignocellulose) is retained when the platform is used at high temperatures (e.g., 85-88°C). Moreover, enhancement persists on acid-pretreated substrates prompting the question: Are HSPs acid tolerant? In this study, HSP structural integrity is examined at different temperatures and pH. Far-UV circular dichroism and intrinsic fluorescence indicate HSPα and HSPβ retain structural integrity at neutral pH over a range of temperatures (25-90°C) while HSPβ-coh is less tolerant to thermal stress. Structural integrity is compromised for all subunits at ultra-low pH (i.e., pH 2) with HSPα showing the most susceptibility. Secondary structures of all HSPs are resilient under mildly acidic conditions (pH 4). ANS binding assays indicate a shift in tertiary structure for all subunits at ultra-low pH. Limited trypsin digestion reveals that the backbone of HSPβ-coh is the most flexible and HSPβ is the most resistant. Results suggest that HSPα and HSPβ are more resilient than HSPβ-coh under thermal challenge and that there are limits to the acid tolerance of all HSPs.