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Hsp33, a prokaryotic redox-regulated holding chaperone, has been recently identified to be able to exhibit an unfoldase and aggregase activity against elongation factor Tu (EF-Tu) in its reduced state. In this study, we investigated the effect of elongation factor Ts (EF-Ts) and trigger factor (TF) on Hsp33-mediated EF-Tu unfolding and aggregation using gel filtration, light scattering, circular dichroism, and isothermal titration calorimetry. We found that EF-Tu unfolding and subsequent aggregation induced by Hsp33 were evident even in its complex state with EF-Ts, which enhanced EF-Tu stability. In addition, although TF alone had no substantial effect on the stability of EF-Tu, it markedly amplified the Hsp33-mediated EF-Tu unfolding and aggregation. Collectively, the present results constitute the first example of synergistic unfoldase/aggregase activity of molecular chaperones and suggest that the stability of EF-Tu is modulated by a sophisticated network of molecular chaperones to regulate protein biosynthesis in cells under stress conditions.

β2-microglobulin (β2m), the light chain of the MHC-I complex, is associated with dialysis-related amyloidosis (DRA). Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m variant, which showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Here, we investigated the role of the D76N mutation in the amyloid formation of β2m by point mutations affecting the Asp76-Lys41 ion-pair of WT β2m and the charge cluster on Asp38. Using a variety of biophysical techniques, we investigated the conformational stability and partial unfolding of the native state of the variants, as well as their amyloidogenic propensity and the stability of amyloid fibrils under various conditions. Furthermore, we studied the intermolecular interactions of WT and mutant proteins with various binding partners that might have in vivo relevance. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the deleterious synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules (e.g., lipids) and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular components, including extracellular matrix proteins.

P5 is one of protein disulfide isomerase family proteins (PDIs) involved in endoplasmic reticulum (ER) protein quality control that assists oxidative folding, inhibits protein aggregation, and regulates the unfolded protein response. P5 reportedly interacts with other PDIs via intermolecular disulfide bonds in cultured cells, but it remains unclear whether complex formation between P5 and other PDIs is involved in regulating enzymatic and chaperone functions. Herein, we established the far-western blot method to detect non-covalent interactions between P5 and other PDIs and found that PDI and ERp72 are partner proteins of P5. The enzymatic activity of P5-mediated oxidative folding is up-regulated by PDI, while the chaperone activity of P5 is stimulated by ERp72. These findings shed light on the mechanism by which the complex formations among PDIs drive to synergistically accelerate protein folding and prevents aggregation. This knowledge has implications for understanding misfolding-related pathology.

Oxygen is a key atom that maintains biomolecular structures, regulates various physiological processes, and mediates various biomolecular interactions. Oxygen-17 (17O), therefore, has been proposed as a useful probe that can provide detailed information about various physicochemical features of proteins. This is attributed to the facts that (1) 17O is an active isotope for nuclear magnetic resonance (NMR) spectroscopic approaches; (2) NMR spectroscopy is one of the most suitable tools for characterizing the structural and dynamical features of biomolecules under native-like conditions; and (3) oxygen atoms are frequently involved in essential hydrogen bonds for the structural and functional integrity of proteins or related biomolecules. Although 17O NMR spectroscopic investigations of biomolecules have been considerably hampered due to low natural abundance and the quadruple characteristics of the 17O nucleus, recent theoretical and technical developments have revolutionized this methodology to be optimally poised as a unique and widely applicable tool for determining protein structure and dynamics. In this review, we recapitulate recent developments in 17O NMR spectroscopy to characterize protein structure and folding. In addition, we discuss the highly promising advantages of this methodology over other techniques and explain why further technical and experimental advancements are highly desired.