Stability Comparisons between Natural Archaeal and Engineered Archaeal‐Bacterial Heat‐Shock Protein Subunits (α, β, and β‐cohesin) and their Oligomeric Complexes
Authors: Sreenivasulu Basha , Gundampati Ravi Kumar , Furr Mercede , Agrawal Shilpi , Alraawi Zeina , Thallapuranam Suresh Kumar , Ceballos Ruben Michael 
Published: April 2020

Hyperthermophilic archaea of the Order Sulfolobales express group II chaperonins, which are commonly referred to as “heat‐shock proteins” (HSPs) due to their upregulated expression upon heat shock. There are three HSP subtypes (HSPα, HSPβ, and HSPγ). Of these, HSPα and HSPβ (a.k.a., TF55 and TF56) subunits are the most thoroughly characterized and are known to stoichiometrically assemble into double‐nonameric (18‐mer) ring structures. These HSP complexes are known to play a significant role not only in the folding of nascent proteins but also in the protection of cytosolic proteins within cells under environmental stress such as high temperature flux. In this context, there has been significant interest in re‐engineering the system as an enzyme protection platform for industrial applications. The first such prototype platform was called a “rosettasome”. The rosettasome is an 18‐mer complex composed of subunits that were fusion proteins of the archaeal HSPβ and cohesion (Type I) from Clostridium thermocellum. Enzymes endowed with a dockerin (Type I) domain bind to cohesion and consequently sequester enzymes to the HSP complex. We previously demonstrated that use of cellulase‐charged rosettasomes on acid (or alkaline) pretreated wheat straw (and corn pericarp) results in an increase in lignocellulose deconstruction and polysaccharide reduction to simple fermentable sugars (e.g., glucose). However, the two‐fold enhancement observed was not sufficient to render the system economically viable. Recently, we re‐engineered the HSP system into a next‐generation mobile enzyme sequestration platform (mESP). In this study, the relative stability of HSP subunits (natural and engineered) and HSP complexes with different subunit compositions are assessed to determine the mESP configuration that exhibits the greatest thermo‐/acido‐tolerance. Transmission electron micrographs reveal the formation of both nonameric ring and nonameric double‐ring (18‐mer) structures. Using differential scanning calorimetry (DSC), the thermodynamic stability of individual subunits as well as homomeric and heteromeric HSP complexes were examined. Results from analytical ultracentrifugation (in conjunction with DSC, TEM, and gel electrophoresis data), reveal the composition and relative stability of HSP complexes. These data show that HSPα has the highest thermal stability of the three subunits tested. In addition, these data indicate that stability of HSP complexes is significantly dependent on the conditions used in complex assembly and subunit stoichiometry. These results and implications for mESP technology are discussed in detail.Support or Funding InformationU.S. National Science Foundation Molecular and Cellular Biosciences (MCB) grant (Award No. 1818346; PI‐Ceballos); U.S. National Science Foundation EArly‐concept Grants for Exploratory Research (EAGER) grant (Award No. 0929484; PI‐Ceballos)