The Chaperonin GroEL: A Versatile Tool for Applied Biotechnology Platforms

Abstract

The nucleotide-free chaperonin GroEL is capable of capturing transient unfolded or partially unfolded states that flicker in and out of existence due to large-scale protein dynamic vibrational modes. In this work, three short vignettes are presented to highlight our continuing advances in the application of GroEL biosensor biolayer interferometry (BLI) technologies and includes expanded uses of GroEL as a molecular scaffold for electron microscopy determination. The first example presents an extension of the ability to detect dynamic pre-aggregate transients in therapeutic protein solutions where the assessment of the kinetic stability of any folded protein or, as shown herein, quantitative detection of mutant-type protein when mixed with wild-type native counterparts. Secondly, using a BLI denaturation pulse assay with GroEL, the comparison of kinetically controlled denaturation isotherms of various von Willebrand factor (vWF) triple A domain mutant-types is shown. These mutant-types are single point mutations that locally disorder the A1 platelet binding domain resulting in one gain of function and one loss of function phenotype. Clear, separate, and reproducible kinetic deviations in the mutant-type isotherms exist when compared with the wild-type curve. Finally, expanding on previous electron microscopy (EM) advances using GroEL as both a protein scaffold surface and a release platform, examples are presented where GroEL-protein complexes can be imaged using electron microscopy tilt series and the low-resolution structures of aggregation-prone proteins that have interacted with GroEL. The ability of GroEL to bind hydrophobic regions and transient partially folded states allows one to employ this unique molecular chaperone both as a versatile structural scaffold and as a sensor of a protein's folded states.

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro

“…The protein synthesis machinery may produce aberrantly folded proteins prone to aggregation or rapid intracellular degradation. Therefore, cells invest in a complex network of molecular chaperones, which provide constant surveillance for protein homeostasis; owing to the great capacity of these molecular machines, the GroE chaperonin system is successfully employed in protein refolding strategies and biotechnological applications ( 30 , 31 ).…”

More than a simple epithelial layer: multifunctional role of echinoderm coelomic epithelium

“…Several chaperons, like endoplasmic reticulum chaperone BiP, are members of the endoplasmic reticulum quality-control system tasked with detecting proteins that fail to mature properly and to target them for cytosolic degradation (Pobre et al 2019). Otherwise, misfolded or partially unfolded proteins are recognized by the GroE chaperonin system that directs them to continue the folding process (O'Neil et al 2018). Other factors intervening in the folding process are the FK506 binding proteins (FKBPs) and cyclophilin B, members of a large family of proteins that possess peptidyl prolyl cis/trans isomerase (PPIase) domains, which alter the conformation of target * Proteins related to exosomal processes proteins (Tong and Jiang 2015;Khong et al 2020).…”

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties

“…The intermediate domains act as hinges between apical and equatorial domains, providing allosteric conformational changes [ 8 , 13 , 19 ]. The functional and structural peculiarities of chaperonins are of importance due to their potential usage in biotechnology and biomedicine projects [ 18 , 20 , 21 , 22 , 23 ]. Some investigators used fusions within the GroEL cavity to produce toxic or prone-to-aggregation polypeptides [ 21 , 22 ], while others employed the tight binding of hydrophobic drugs by GroEL apical domains for the delivery of these drugs to tumors [ 18 ].…”