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bioRxiv · 10.1101/2021.08.21.457208

Stability Comparisons between Natural versus Engineered Archaeal Heat Shock Proteins

Furr, M. M.·Basha, S.·Agrawal, S.·Alraawi, Z.·Ghosh, P.·Stacy, C. L.·Suresh Kumar, T. K.·Ceballos, R. M.
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Abstract

Crenarchaeal group II chaperonins (a.k.a., "heat shock" proteins, HSPs) are abundantly expressed in species of the family Sulfolobaceae. HSP and HSP{beta} 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. HSP{beta}-coh, a fusion protein comprised of HSP{beta} 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{degrees}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 HSP and HSP{beta} retain structural integrity at neutral pH over a range of temperatures (25-90{degrees}C) while HSP{beta}-coh is less tolerant to thermal stress. Structural integrity is compromised for all subunits at ultra-low pH (i.e., pH 2) with HSP 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 HSP{beta}-coh is the most flexible and HSP{beta} is the most resistant. Results suggest that HSP and HSP{beta} are more resilient than HSP{beta}-coh under thermal challenge and that there are limits to the acid tolerance of all HSPs.

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Furr, M. M., Basha, S., Agrawal, S., Alraawi, Z., Ghosh, P., Stacy, C. L., Suresh Kumar, T. K., Ceballos, R. M.. 2021-08-22. Stability Comparisons between Natural versus Engineered Archaeal Heat Shock Proteins. https://doi.org/10.1101/2021.08.21.457208

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