An “Onion‐like” Model of Protein Unfolding: Collective versus Site Specific Approaches

Abstract

Approximating protein unfolding by an all-or-none cooperative event is a convenient assumption that can provide precious global information on protein stability. It is however quickly emerging that the scenario is far more complex and that global denaturation curves often hide a rich heterogeneity of states that are largely probe dependent. In this review, we revisit the importance of gaining site-specific information on the unfold-ing process. We focus on nuclear magnetic resonance, as this is the main technique able to provide site-specific information. We review historical and most modern approaches that have allowed an appreciable advancement of the field of protein folding. We also demonstrate how unfolding is a reporter dependent event, suggesting the outmost importance of selecting the reporter carefully.

Monitoring protein unfolding transitions by NMR-spectroscopy

“…a crowded cellular environment, liquid-liquid phase separation or high pressures. As also pointed out in an recent review about NMR detected protein unfolding (Politou et al 2021) it is not enough to interpret unfolding transitions or protein stability data of single residue but the analysis of all accessible sites will give a global picture about which core residues form the cooperative folding unit and which residues experience deviating local effects. These deviations should not per se lead to the substitution of a simple two-state folding model by a more complex one.…”

“…Local interactions with the denaturant and thus induced noncooperative conformational changes might occur and can be easily identified by deviating from the global unfolding transition. Several examples are discussed in a recent review (Politou et al 2021). In these cases the concept of cooperative folding still holds, since a cooperative folding unit exists and on top local effects can be identified and interpreted.…”

Unfolding under Pressure: An NMR Perspective

“…Initial in vitro studies, both using temperature and pressure perturbations, were performed with methods addressing global structural features, that is, the whole secondary and/or tertiary structure, for instance using circular dichroism spectroscopy or intrinsic fluorescence [6] . The possibility of using nuclear magnetic resonance spectroscopy has extended unfolding studies to the behaviour of individual residues, providing a sequence‐specific map of local unfolding [7,8] .…”

“…[5] Initial in vitro studies, both using temperature and pressure perturbations, were performed with methods addressing global structural features, that is, the whole secondary and/or tertiary structure, for instance using circular dichroism spectroscopy or intrinsic fluorescence. [6] The possibility of using nuclear magnetic resonance spectroscopy has extended unfolding studies to the behaviour of individual residues, providing a sequencespecific map of local unfolding. [7,8] While protein unfolding has widely been analysed by NMR at high and, in more recent times, at low temperature, [9,10] still relatively few studies have addressed pressure unfolding by NMR, probably because of the technical difficulties to obtain this transition, plus the fact that the topic is often considered only a niche field, with relatively little direct interest for biological and/or biotechnological applications.…”