Stacking of nanorings to generate nanotubes for acceleration of protein refolding

Refolding of denatured proteins effectively occurred simultaneously with release from a long and narrow nanotube, which is formed by uniaxial stacking of nanorings.

Escherichia coli small heat shock protein IbpA is an aggregation-sensor that self-regulates its own expression at a translational level

Aggregation is an inherent characteristic of proteins. Risk management strategies to reduce aggregation are critical for cells to survive upon stresses that induce aggregation. Cells cope with protein aggregation by utilizing a variety of chaperones, as exemplified by heat-shock proteins (Hsps). The heat stress-induced expression of IbpA and IbpB, small Hsps in Escherichia coli, is regulated by the σ32 heat-shock transcriptional regulator and the temperature-dependent translational regulation via mRNA heat fluctuation. We found that, even without heat stress, either the expression of aggregation-prone proteins or the ibpA gene deletion profoundly increases the expression of IbpA. Combined with other evidence, we propose novel mechanisms for the regulation of the small Hsp expression. Oligomeric IbpA self-represses the ibpA/ibpB expression at the translational level, but the self-repression is relieved by the sequestration of IbpA into protein aggregates. Thus, the function of IbpA as a chaperone to form co-aggregates is harnessed as an aggregation sensor to tightly regulate the IbpA level. Since the excessive preemptive supply of IbpA in advance of stress is harmful, the prodigious and rapid expression of IbpA/IbpB on demand is necessary for IbpA to function as a first line of defense against acute protein aggregation.Author summaryAll organisms have protein quality control systems against stresses disturbing cellular protein homeostasis (proteostasis). The systems have multiple stages: folding, degradation, and sequestration. Sequestration of denatured proteins is the first step to support other maintenance strategies. Small heat shock proteins (sHsps), which are well-conserved chaperones, are representative “sequestrases” that co-aggregate with denatured proteins. We found that IbpA, an Escherichia coli sHsp, is a direct mediator for negative feedback regulation at the translational level. Recruitment of IbpA into the protein aggregates relieves the ibpA expression suppression. This novel mechanism of IbpA as an aggregation-sensor tightly regulates the IbpA level, enabling the sHsp to function as a sequestrase upon aggregation stress.

Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution

The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL. Native electrophoresis has demonstrated that these proteins tend to be released from the GroEL-GroES complex. With the use of biotin- and fluorescein-labeled denatured proteins and streptavidin fused with luciferase aequorin (the so-called streptavidin trap), the presence of denatured proteins in bulk solution after GroES and Mg-ATP addition has been confirmed. The time of GroES-induced dissociation of a denatured protein from the GroEL surface was estimated using the stopped-flow technique and found to be much shorter than the proposed time of the GroEL ATPase cycle.

A method to measure the denatured proteins in the corona of nanoparticles based on the specific adsorption of Hsp90ab1

The specific adsorption of Hsp90ab1 is exploited to assess the protein denaturation in the biological corona of nanoparticles.