Cellular and Molecular Life Sciences (2018) 75:3907–3929 https://doi.org/10.1007/s00018-018-2894-9 Cellular andMolecular Life Sciences REVIEW Intrinsically disordered proteins in crowded milieu: when chaos prevails within the cellular gumbo Alexander V. Fonin1 · April L. Darling2 · Irina M. Kuznetsova1 · Konstantin K. Turoverov1,3 · Vladimir N. Uversky2 Received: 8 June 2018 / Revised: 24 July 2018 / Accepted: 26 July 2018 / Published online: 31 July 2018 © Springer Nature Switzerland AG 2018 Abstract Efects of macromolecular crowding on structural and functional properties of ordered proteins, their folding, interactability, and aggregation are well documented. Much less is known about how macromolecular crowding might afect structural and functional behaviour of intrinsically disordered proteins (IDPs) or intrinsically disordered protein regions (IDPRs). To fll this gap, this review represents a systematic analysis of the available literature data on the behaviour of IDPs/IDPRs in crowded environment. Although it was hypothesized that, due to the excluded-volume efects present in crowded environments, IDPs/IDPRs would invariantly fold in the presence of high concentrations of crowding agents or in the crowded cellular environment, accumulated data indicate that, based on their response to the presence of crowders, IDPs/IDPRs can be grouped into three major categories, foldable, non-foldable, and unfoldable. This is because natural cellular environment is not simply characterized by the presence of high concentration of “inert” macromolecules, but represents an active milieu, components of which are engaged in direct physical interactions and soft interactions with target proteins. Some of these interactions with cellular components can cause (local) unfolding of query proteins. In other words, since crowding can cause both folding and unfolding of an IDP or its regions, the outputs of the plac- ing of a query protein to the crowded environment would depend on the balance between these two processes. As a result, and because of the spatio-temporal heterogeneity in structural organization of IDPs, macromolecular crowding can diferently afect structures of diferent IDPs. Recent studies indicate that some IDPs are able to undergo liquid–liquid-phase transitions leading to the formation of various proteinaceous membrane-less organelles (PMLOs). Although interiors of such PMLOs are self-crowded, being characterized by locally increased concentrations of phase-separating IDPs, these IDPs are minimally foldable or even non- foldable at all (at least within the physiologically safe time-frame of normal PMLO existence). Keywords Intrinsically disordered protein · Intrinsically disordered protein region · Macromolecular crowding · Proteinaceous membrane-less organelles · Conformational stability · Induced folding Alexander V. Fonin and April L. Darling contributed equally to this work. * Vladimir N. Uversky 1 Laboratory of Structural Dynamics, Stability and Folding [email protected] of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russian Federation Alexander V. Fonin [email protected] 2 Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College April L. Darling of Medicine, University of South Florida, Tampa, FL, USA [email protected] 3 St. Petersburg State Polytechnical University, St. Petersburg, Irina M. Kuznetsova Russian Federation [email protected] Konstantin K. Turoverov [email protected] Vol.:(0123456789)1 3 3908 A. V. Fonin et al. Introduction these observations, an important conclusion was made that purely excluded-volume efects from macromolecules are Macromolecular crowding and complexity more complex than commonly assumed (and potentially are of the cellular environment much smaller than generally believed), it was also pointed out that high concentrations of macromolecules in the cell Cell interior is known to be extremely crowded due to serve as a foundation of the signifcant non-ideality [29]. the presence of high concentrations (up to 400 mg/ml) of Therefore, a signifcant caution should be used while using various biological macromolecules, such as carbohydrates, high concentrations of “inert” macromolecules (which, in nucleic acids, and proteins [1]. This crowded environment fact, can make specifc interactions with proteins and nucleic [2, 3] imposes considerable restrictions on the amount of acids) in experiments in vitro to avoid introduction of unan- free water [1, 2, 4–7], decreases accessible volume (since ticipated intersolute interactions [29]. macromolecules occupy a signifcant fraction of the cellular Although, in recent years, the signifcance of macromo- volume (typically 20–30%), the volume occupied by these lecular crowding as important but mostly neglected vari- solutes is unavailable to other molecules, because two mol- able in biochemical studies is gaining attention [4, 6], the ecules cannot be in the same place at the same time) [4, 8], principle shortcoming of a typical test-tube experiment causes confnement [9], as well as afects solvent viscosity, dedicated to the analysis of a biological macromolecule or thereby modulating intracellular difusion [10–12]. Recent a biochemical reaction continues to be a failure to acknowl- study, where the conditions of macromolecular crowding edge this phenomenon. In fact, macromolecular crowd- were modelled by solutions containing high concentrations ing is not typically included as a constituent of standard of a model “crowding agent” polyethylene glycol, revealed “physiological conditions”, and the vast majority of in vitro that the crowder is also able to induce noticeable changes in experiments are usually performed under the relatively ideal the solvent properties of water, such as solvent dipolarity/ thermodynamic conditions of low protein and moderate salt polarizability, hydrogen-bond donor acidity, and hydrogen- concentrations. One of the reasons for this negligence is the bond acceptor basicity [13]. lack of general understanding of what should be looked at Thermodynamically, the efects of the crowded environ- (confnement, excluded volume, changed solvent properties ment on chemical reactions are attributed to the excluded- of water, or altered viscosity) while designing the conditions volume concept [2–4, 8, 9]. It was pointed out that the that would appropriately model biological fuids. Tradition- crowded environment of biological fuids, and especially ally, the efect of excluded volume on the behaviour of query related excluded-volume efects, may have signifcant infu- compounds is examined experimentally using concentrated ence on conformational stability and structure of biologi- solutions of some crowding agents, such as polymers (e.g., cal macromolecules [3, 14–17] and alter macromolecular polyethylene glycol, Ucon, polyvinylpyrrolidone, dextran, equilibrium, afecting protein folding [18–20], efciency of Ficoll, etc.) or highly soluble “inert” proteins (e.g., lysozyme various biological reactions [4, 18, 19], binding of small or serum albumin) [8, 22]. Crowded cellular environment molecules, enzymatic activity, protein–nucleic acid inter- alters molecular difusion, and the corresponding efects of actions, protein–protein interactions [3, 21], and rates and such environment on the translational mobility can also be extent of protein aggregation and amyloid fbril formation modelled by synthetic crowders, concentration, and size of [22–24]. Furthermore, the efciency of excluded-volume which can modulate probe microviscosity [30]. As far as efects posed by a crowder is known to be dependent on modelling of the confned intracellular space is concerned, the relative hydrodynamic dimensions of crowder and tar- one of the related approaches includes encapsulation of a get molecule (crowdee), with the strongest efects being target protein in silica glass using the sol–gel techniques to ascribed to a situation, where the crowder and the crowdee create a crowded microenvironment [31, 32]. Although the have comparable hydrodynamic volumes [25–28]. However, fraction of the total volume excluded by the silica matrix is when the crowder volume becomes too large, larger “caves” lower than the fractional volume occupied by macromol- will be formed between the crowder molecules that could ecules in living cell [14, 15], the size of protein-occupied accumulate more than one molecule of crowdee. In other pores in these gels has the same order of magnitude as the words, although globally accessible volume will decrease diameter of protein [31]. Furthermore, solvents can easily in the presence of such large crowder, locally accessible permeate silica matrix due to the porosity of the resulting volume might increase [25]. Recently, it was pointed out by glass, whereas the encapsulated macromolecules cannot gen- Kim Sharp that when the steric efects of macromolecular erally escape [14]. This sol–gel glass encapsulation was used crowders and small molecules, such as ions, are treated on for the analysis of several proteins at the variety of solvent an equal footing, small molecules act as more efcient crow- conditions [33–36]. ders than the large macromolecules [29]. Although based on One should keep in mind that the potential efects of cel- lular environment on structural properties, conformational 1 3 Intrinsically disordered proteins in crowded milieu: when chaos prevails within the cellular… 3909 behaviour, and functionality of proteins are much more Golgi apparatus, lysosomes, nucleus,