Crowding revisited: Open questions and future perspectives.

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°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.

Molecular Crowding: The History and Development of a Scientific Paradigm.

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.

The Wide World of Coacervates: From the Sea to Neurodegeneration.

The formation of immiscible liquid phases or coacervates is a phenomenon widely observed in biology. Marine organisms, for instance, use liquid-liquid phase separation (LLPS) as the precursor phase to form various fibrillar or crustaceous materials that are essential for surface adhesion. More recently, the importance of LLPS has been realized in the compartmentalization of living cells and in obtaining ordered but dynamic partitions that can be reversed according to necessity. Here, we compare the properties, features, and peculiarities of intracellular and extracellular coacervates, drawing parallels and learning from the differences. A more general view of the phenomenon may in the future inform new studies to allow a better comprehension of its laws.

Unfolding under Pressure: An NMR Perspective.

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.

Man does not live by intrinsically unstructured proteins alone: The role of structured regions in aggregation.

Protein misfolding is a topic that is of primary interest both in biology and medicine because of its impact on fundamental processes and disease. In this review, we revisit the concept of protein misfolding and discuss how the field has evolved from the study of globular folded proteins to focusing mainly on intrinsically unstructured and often disordered regions. We argue that this shift of paradigm reflects the more recent realisation that misfolding may not only be an adverse event, as originally considered, but also may fulfil a basic biological need to compartmentalise the cell with transient reversible granules. We nevertheless provide examples in which structure is an important component of a much more complex aggregation behaviour that involves both structured and unstructured regions of a protein. We thus suggest that a more comprehensive evaluation of the mechanisms that lead to aggregation might be necessary.

The seesaw between normal function and protein aggregation: How functional interactions may increase protein solubility.

Protein aggregation has been studied for at least 3 decades, and many of the principles that regulate this event are relatively well understood. Here, however, we present a different perspective to explain why proteins aggregate: we argue that aggregation may occur as a side-effect of the lack of one or more natural partners that, under physiologic conditions, would act as chaperones. This would explain why the same surfaces that have evolved for functional purposes are also those that favour aggregation. In the course of reviewing this field, we substantiate our hypothesis with three paradigmatic examples that argue for the generality of our proposal. An obvious corollary of this hypothesis is, of course, that targeting the physiological partners of a protein could be the most direct and specific approach to designing anti-aggregation molecules. Our analysis may thus inform a different strategy for combating diseases of protein aggregation and misfolding.

Heat and cold denaturation of yeast frataxin: The effect of pressure.

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.

The Protein Unfolded State: One, No One and One Hundred Thousand.

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.

Recipes for Inducing Cold Denaturation in an Otherwise Stable Protein.

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.

An "Onion-like" Model of Protein Unfolding: Collective versus Site Specific Approaches.

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.

RNA as the stone guest of protein aggregation.

The study of prions as infectious aggregates dates several decades. From its original formulation, the definition of a prion has progressively changed to the point that many aggregation-prone proteins are now considered bona fide prions. RNA molecules, not included in the original 'protein-only hypothesis', are also being recognized as important factors contributing to the 'prion behaviour', that implies the transmissibility of an aberrant fold. In particular, an association has recently emerged between aggregation and the assembly of prion-like proteins in RNA-rich complexes, associated with both physiological and pathological events. Here, we discuss the historical rising of the concept of prion-like domains, their relation to RNA and their role in protein aggregation. As a paradigmatic example, we present the case study of TDP-43, an RNA-binding prion-like protein associated with amyotrophic lateral sclerosis. Through this example, we demonstrate how the current definition of prions has incorporated quite different concepts making the meaning of the term richer and more stimulating. An important message that emerges from our analysis is the dual role of RNA in protein aggregation, making RNA, that has been considered for many years a 'silent presence' or the 'stone guest' of protein aggregation, an important component of the process.

Generalized View of Protein Folding: In Medio Stat Virtus.

Proteins are often described in textbooks as being only "marginally stable" but many proteins, specifically those with a high free energy of unfolding are, in fact, so stable that they exist only in the fully folded state except under harsh denaturing conditions. Proteins that are truly only marginally stable, those with a low free energy of unfolding, exist as an equilibrium mixture of folded and unfolded forms under "normal" conditions. To some extent such proteins have some features in common with "intrinsically disordered" proteins. We analyzed the relationship between these marginally stable proteins and intrinsically disordered proteins in order to fully understand the twilight zone that distinguishes the two ensembles in the hope of clarifying the fuzzy borders of the current classification that divides the protein world into folded and intrinsically disordered ones. Our analysis suggests that the division may be too drastic and misleading, because it puts within the same category proteins with very different behaviors. We propose a restricted, albeit operational, definition of "marginally stable proteins", referring by this term only to proteins whose free energy difference between the folded and unfolded states falls in the interval 0-3 kcal/mol. These proteins have special features because they normally exist as equilibrium mixtures of folded and unfolded species or as molten globule states. This coexistence makes marginally stable proteins ideal tools to study even small environmental changes to which they may behave as natural sensors.

The Emperor's new clothes: Myths and truths of in-cell NMR.

In-cell NMR is a technique developed to study the structure and dynamical behavior of biological macromolecules in their natural environment, circumventing all isolation and purification steps. In principle, the potentialities of the technique are enormous, not only for the possibility of bypassing all purification steps but, even more importantly, for the wealth of information that can be gained from directly monitoring interactions among biological macromolecules in a natural cell. Here, we review critically the promises, successes and limits of this technique as it stands now. Interestingly, many of the problems of NMR in bacterial cells stem from the artificially high concentration of the protein under study whose overexpression is anyway necessary to select it from the background. This has, as a consequence, that when overexpressed, most globular proteins, do not show an NMR spectrum, limiting the applicability of the technique to intrinsically unfolded or specifically behaving proteins. The outlook for in-cell NMR of eukaryotic cells is more promising and is possibly the most attracting aspect for the future.

Cold Denaturation Unveiled: Molecular Mechanism of the Asymmetric Unfolding of Yeast Frataxin.

What is the mechanism that determines the denaturation of proteins at low temperatures, which is, by now, recognized as a fundamental property of all proteins? We present experimental evidence that clarifies the role of specific interactions that favor the entrance of water into the hydrophobic core, a mechanism originally proposed by Privalov but never proved experimentally. By using a combination of molecular dynamics simulation, molecular biology, and biophysics, we identified a cluster of negatively charged residues that represents a preferential gate for the entrance of water molecules into the core. Even single-residue mutations in this cluster, from acidic to neutral residues, affect cold denaturation much more than heat denaturation, suppressing cold denaturation at temperatures above zero degrees. The molecular mechanism of the cold denaturation of yeast frataxin is intrinsically different from that of heat denaturation.

Sweet, bitter and umami receptors: a complex relationship.

Sweet and bitter are taste qualities linked to food acceptance and rejection in humans. It was long thought that these taste sensations were closely related, but the discovery and characterization of taste receptors revealed that mammals express a single sweet receptor and many unrelated bitter receptors. Bitter-tasting chiral isomers of sweet compounds can bind to the umami receptor, rather than bitter receptors, and elicit the bitter sensation through crosstalk between labelled cells. In support of crosstalk between labelled cells, recent findings suggest that, although most receptor taste cells respond to only one taste, most presynaptic taste cells accept signals from labelled cells that respond to two or more different taste qualities.

Revitalizing an important field in biophysics: The new frontiers of molecular crowding.

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.

A natural and readily available crowding agent: NMR studies of proteins in hen egg white.

In vitro studies of biological macromolecules are usually performed in dilute, buffered solutions containing one or just a few different biological macromolecules. Under these conditions, the interactions among molecules are diffusion limited. On the contrary, in living systems, macromolecules of a given type are surrounded by many others, at very high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides, or even synthetic polymers. Here, we propose the use of hen egg white (HEW) as a simple natural medium, with all features of the media of crowded cells, that could be used by any researcher without difficulty and inexpensively. We present a study of the stability and dynamics behavior of model proteins in HEW, chosen as a prototypical, readily accessible natural medium that can mimic cytosol. We show that two typical globular proteins, dissolved in HEW, give NMR spectra very similar to those obtained in dilute buffers, although dynamic parameters are clearly affected by the crowded medium. The thermal stability of one of these proteins, measured in a range comprising both heat and cold denaturation, is also similar to that in buffer. Our data open new possibilities to the study of proteins in natural crowded media.

Determinants of sweetness in proteins: a topological approach.

Sweet taste in mammals is accounted for by a single receptor that shares homology with a metabotropic glutamate receptor. Most sweeteners are small molecular weight molecules that interact with small cavities in the so-called Venus Flytrap domains of the sweet receptor. The mechanism of action of larger molecules such as sweet proteins is, however, more difficult to interpret. The first and still the only general mechanism proposed for the action of sweet proteins, the "wedge model," hypothesizes that proteins bind to an external binding site of the active conformation of the sweet receptor. Here, I have extended the concept that inspired the wedge model using a combination of structural analysis, bioinformatics tools, and a relatively large dataset of mutations of the two most extensively studied sweet proteins, monellin and brazzein. I show here that it is possible to single out, among the ensemble yielded by low-resolution docking, a unique complex that satisfies simple topological constraints. These models of the complexes of monellin and brazzein are fully consistent with experimental evidence, thus providing predicting power for further validation of the wedge model.

Striking Dependence of Protein Sweetness on Water Quality: The Role of the Ionic Strength.

Sweet proteins are the sweetest natural molecules. This aspect prompted several proposals for their use as food additives, mainly because the amounts to be added to food would be very small and safe for people suffering from sucrose-linked diseases. During studies of sweet proteins as food additives we found that their sweetness is affected by water salinity, while there is no influence on protein's structure. Parallel tasting of small size sweeteners revealed no influence of the water quality. This result is explained by the interference of ionic strength with the mechanism of action of sweet proteins and provides an experimental validation of the wedge model for the interaction of proteins with the sweet receptor.