This chapter is dedicated to analysing how biological nuclear magnetic resonance (NMR) has evolved in the more than 70 years from its discovery and to discussing some of the applications of this technique to biological systems. To restrict a vast field, we treat liquid-state NMR leaving out solid-state applications that, although very important, are less consolidated and still fast evolving. We focus in particular on studies in complex environments, such as in-cell and/or crowded environments, that is a subject in which NMR can give its very best. We provide examples of various applications and hopefully show how versatile the technique is. We also briefly discuss how NMR can be coupled with other techniques to expand its horizons, increasing the intrinsic potentialities of this powerful technique.

The conformational transition related to protein unfolding is often considered as the transition between the folded conformation and one unfolded form. However, there are many aspects that hint at a much richer and more complex unfolded state made of a conformational ensemble not only for the unfolded species but also during the unfolding process. Proteins often sample intermediate conformations that may retain elements of secondary structure, preserve part of the hydrophobic core, or display localized unfolding restricted to specific regions. During the unfolding pathway, there are often minor transitions involving local secondary-structure regions outside the hydrophobic core. Many researchers have also hypothesized the existence of one or more intermediates, albeit usually invisible because they are low populated. In the present Mini-Review, we re-examine critically crucial aspects of this fascinating problem starting from our own experience in protein stability and unfolding and revise the limitations and implications of the two-state model.

The present work proposes an explanation for a recent observation that has conclusively proven that heavy water, that is, water containing the non-radioactive isotope of hydrogen deuterium, is mildly sweet, at variance with the tasteless common water. No firm explanation was proposed for this unexpected behavior. Yet, the subject is far from being an irrelevant curiosity, as the explanation of yet unidentified properties of the sweet receptor can help us to understand the molecular bases of food appreciation that have direct repercussions on pathologies such as diabetes and obesity. Here, a simple but convincing structural explanation of the taste of heavy water is proposed that is based on the influence of heavy water on the conformation of the active form of the receptor. The explanation requires the concept of "constitutive receptor activity", that is, a notion well accepted in many areas of pharmacology but clearly neglected in reference to taste receptors. We discuss how constitutive activity also explains other properties such as the recognition of sweet proteins that are several thousand times sweeter than small carbohydrates.

Yeast frataxin (Yfh1) is a small natural protein from yeast that has the unusual property of undergoing cold denaturation at temperatures above the freezing point of water when under conditions of low ionic strength. This peculiarity, together with remarkable resilience, allows the determination, for the whole protein as well as for individual residues, of the stability curve, that is the temperature dependence of the free energy difference between the unfolded and folded forms. The ease of measuring stability curves without the need to add denaturants or introduce ad hoc destabilizing mutations makes this protein an ideal 'tool' for investigating the influence of many environmental factors on protein stability. The present review aims at recapitulating all the open questions that Yfh1 has helped to address, including understanding the differences and commonalities of the cold, heat and pressure unfolded states. This protein thus offers a unique tool for studying aspects of protein stability so far been considered difficult to assess and provides important guidelines that could allow the identification of other similar systems.

Proteins unfold under different environmental insults, among which are heat, cold, high pressure, and chaotropic agents. Understanding the mechanisms that determine unfolding under each of these conditions is an important problem that directly relates to the physical forces that determine the three-dimensional structure of a protein. Here, we studied a residue-specific description of the unfolding transitions of marginally stable yeast protein Yfh1 using high-pressure nuclear magnetic resonance. We compared the cold, heat, and pressure unfolded states and demonstrated what has up to now been only a hypothesis: the pressure-unfolded spectrum at room temperature shares features in common with that at low but not at high temperature and room pressure, suggesting a tighter similarity of the mechanisms and a similar role of hydration in these two processes. By exploring the phase diagram of the protein and mapping unfolding onto the three-dimensional structure of the protein, we also show that the pressure-induced unfolding pathways at low and high temperatures differ, suggesting a synergic mechanism between pressure- and temperature-induced denaturation. Our observations help us to reconstruct the structural events determining unfolding and distinguish the mechanisms that rule the different processes of unfolding.

A few decades ago, it became clear that most biophysical studies were performed on proteins under conditions not corresponding to those found in vivo, thus limiting the generality of at least some of the conclusions. The main difference was identified in the absence, in the dilute solutions typically used, of large amounts of other macromolecules. Solution conditions similar to those found in vivo were dubbed as crowded. The main consequences of crowding were identified as the influence of crowding on protein stability and on alterations of reaction conditions due to changes in protein activity. Initial experimental work to assess the effect of crowding on protein stability was dominated by theoretical estimations that suggested an increase of the unfolding temperatures between 5 and 20 °C. The present chapter examines some of the techniques typically used in these studies in a critical way, exploring the most common methodologies employed to study proteins under crowded conditions. One of the critical problems is the choice of the crowders. There are two categories of commonly used macromolecular crowders, synthetic polymers and common proteins. Synthetic crowders often do not interfere with optical measurements of proteins. When proteins are used as crowder it is necessary to label the protein under study. We demonstrate that studies of protein stability in crowded solutions provide more valuable information when observing the behaviour of marginally stable proteins.

It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.

Proteins unfold under different environmental insults, among which heat, cold, high pressure and chaotropic agents. Understanding the mechanisms that determine unfolding under each of these condition is an important problem that directly relates to the physical forces that determine the three-dimensional structure of a protein. Here, we studied the mechanism of pressure unfolding of the marginally stable yeast protein Yfh1 using high-pressure nuclear magnetic resonance as this is the most appropriate technique to obtain a residue-specific description of the folding/unfolding transitions of a protein. We demonstrate that the pressure-unfolded spectrum shares features in common with that at low but not at high temperature and room pressure, suggesting a tighter similarity between the two processes that could be explained by a similar role of hydration in the process. This is the first time that comparison between the three infolded states could be tested experimentally, and confirms something that up to now has been only suggested as an hypothesis. By recording the phase diagram of the protein, we also show that temperature switches the pressure-induced unfolding pathway suggesting a synergic mechanism between pressure- and temperature-induced denaturation: at moderate pressures, Yfh1 unfolding at low temperature starts at a patch of negatively charged residues that we have previously demonstrated to induce electrostatic frustration and cause cold denaturation. Heat unfolding involves instead a cavity created by insufficient protection of the hydrophobic core. These observations help us to reconstruct the structural events determining unfolding and distinguish the mechanisms that rule the different processes of unfolding. ### Competing Interest Statement The authors have declared no competing interest.

This review aims to analyse the role of solution nuclear magnetic resonance spectroscopy in pressure-induced in vitro studies of protein unfolding. Although this transition has been neglected for many years because of technical difficulties, it provides important information about the forces that keep protein structure together. We first analyse what pressure unfolding is, then provide a critical overview of how NMR spectroscopy has contributed to the field and evaluate the observables used in these studies. Finally, we discuss the commonalities and differences between pressure-, cold- and heat-induced unfolding. We conclude that, despite specific peculiarities, in both cold and pressure denaturation the important contribution of the state of hydration of nonpolar side chains is a major factor that determines the pressure dependence of the conformational stability of proteins.

Taking into account the presence of the crowded environment of a macromolecule has been an important goal of biology over the past 20 years. Molecular crowding affects the motions, stability and the kinetic behaviour of proteins. New powerful approaches have recently been developed to study molecular crowding, some of which make use of the synchrotron radiation light. The meeting “New Frontiers in Molecular Crowding” was organized in July 2022at the European Synchrotron Radiation facility of Grenoble to discuss the new frontiers of molecular crowding. The workshop brought together researchers from different disciplines to highlight the new developments of the field, including areas where new techniques allow the scientists to gain unprecedently expected information. A key conclusion of the meeting was the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives that could let the molecular crowding field flourish further.

Many in vitro studies, in which proteins have been unfolded by the action of a variety of physical or chemical agents, have led to the definition of a folded versus an unfolded state and to the question of what is the nature of the unfolded state. The unstructured nature of this state could suggest that "the" unfolded state is a unique entity which holds true for all kinds of unfolding processes. This assumption has to be questioned because the unfolding processes under different stress conditions are dictated by entirely different mechanisms. As a consequence, it can be easily understood that the final state, generically referred to as "the unfolded state", can be completely different for each of the unfolding processes. The present review examines recent data on the characteristics of the unfolded states emerging from experiments under different conditions, focusing specific attention to the level of compaction of the unfolded species.

Yfh1 is a yeast protein with the peculiar characteristic to undergo, in the absence of salt, cold denaturation at temperatures above the water freezing point. This feature makes the protein particularly interesting for studies aiming at understanding the rules that determine protein fold stability. Here, we present the phase diagram of Yfh1 unfolding as a function of pressure (0.1-500 MPa) and temperature 278-313 K (5-40°C) both in the absence and in the presence of stabilizers using Trp fluorescence as a monitor. The protein showed a remarkable sensitivity to pressure: at 293 K, pressures around 10 MPa are sufficient to cause 50% of unfolding. Higher pressures were required for the unfolding of the protein in the presence of stabilizers. The phase diagram on the pressure-temperature plane together with a critical comparison between our results and those found in the literature allowed us to draw conclusions on the mechanism of the unfolding process under different environmental conditions.

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 unfolding 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.

Although cold denaturation is a fundamental phenomenon common to all proteins, it can only be observed in a handful of cases where it occurs at temperatures above the freezing point of water. Understanding the mechanisms that determine cold denaturation and the rules that permit its observation is an important challenge. A way to approach them is to be able to induce cold denaturation in an otherwise stable protein by means of mutations. Here, we studied CyaY, a relatively stable bacterial protein with no detectable cold denaturation and a high melting temperature of 54 °C. We have characterized for years the yeast orthologue of CyaY, Yfh1, a protein that undergoes cold and heat denaturation at 5 and 35 °C, respectively. We demonstrate that, by transferring to CyaY the lessons learnt from Yfh1, we can induce cold denaturation by introducing a restricted number of carefully designed mutations aimed at destabilizing the overall fold and inducing electrostatic frustration. We used molecular dynamics simulations to rationalize our findings and demonstrate the individual effects observed experimentally with the various mutants. Our results constitute the first example of rationally designed cold denaturation and demonstrate the importance of electrostatic frustration on the mechanism of cold denaturation.

Although biophysical studies have traditionally been performed in diluted solutions, it was pointed out in the late 1990s that the cellular milieu contains several other macromolecules, creating a condition of molecular crowding. How crowding affects protein stability is an important question heatedly discussed over the past 20 years. Theoretical estimations have suggested a 5-20 degrees C effect of fold stabilisation. This estimate, however, is at variance with what has been verified experimentally that proposes only a limited increase of stability, opening the question whether some of the assumptions taken for granted should be reconsidered. The present review critically analyses the causes of this discrepancy and discusses the limitations and implications of the current concept of crowding.

EDITORIAL article Front. Mol. Biosci., 07 October 2022Sec. Structural Biology Volume 9 - 2022 | https://doi.org/10.3389/fmolb.2022.1020473

AbstractMost techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.

Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.