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How Cooperative Are Protein Folding and Unfolding Transitions?

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How Cooperative Are Protein Folding and Unfolding Transitions? REVIEW How cooperative are protein folding and unfolding transitions? Pooja Malhotra and Jayant B. Udgaonkar* National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru 560065, India Received 26 July 2016; Accepted 9 August 2016 DOI: 10.1002/pro.3015 Published online 13 August 2016 proteinscience.org Abstract: A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and inter- converting by cooperative two-state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computa- tional approaches has demonstrated entirely non-cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative ver- sus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier-less “downhill” folding, as well as for continuous “uphill” unfolding transitions, indicate that gradual non-cooperative processes may be ubiquitous features on the free energy landscape of protein folding. Keywords: cooperativity; intermediates; one-state; population distributions; downhill folding; uphill unfolding Introduction proteins appear to undergo completely cooperative The simplest possible way of describing the transi- folding and unfolding, as their folding reactions are tions between the native (N) and unfolded (U) states found to meet both the kinetic and the thermody- 1–3 of a protein is the two-state U$N model.1 Many namic criteria for two-state folding. This decep- tively simple depiction of protein folding has had Grant sponsors: JC Bose National Research Fellowship, Gov- great practical value. The two-state model allows an ernment of India, Tata Institute of Fundamental Research, the easy determination of the effects of mutations on the Department of Science and Technology, and the Department of Biotechnology, Government of India. stabilities of the transition state (TS) and the N state, with respect to the U state, and enables indi- *Correspondence to: Jayant B. Udgaonkar, National Centre for Biological Sciences, Tata Institute of Fundamental Research, rect inferences to be made about the structure of the Bengaluru 560065, India. E-mail: [email protected] TS and its possible role as a nucleus in protein 1924 PROTEIN SCIENCE 2016 VOL 25:1924—1941 Published by Wiley-Blackwell. VC 2016 The Protein Society folding reactions.4,5 The criteria for two-state folding This review examines the extent of cooperativity are, however, also met by a multi-state folder whose inherent to folding and unfolding transitions, includ- folding reaction is populated by intermediates that ing downhill folding and uphill unfolding. The tun- are too unstable to be detected experimentally.6,7 In able nature as well as the structural basis of many cases, high energy intermediates can be stabi- cooperativity on the free energy landscape of protein lized by appropriate experimental manipulation,8–10 folding has been discussed. Early folding transitions and thereby become detectable. Nevertheless, the that occur prior to the major structure-forming reac- utility of two-state models is so great, that even tions,69 as well as insights gained from theoretical when the folding kinetics may be more correctly and computational approaches70,71 are not discussed describable as multi-state, the two-state model is here, as they have been reviewed elsewhere. invariably extended to account for the kinetics. Multi-step folding is undeniable for many pro- Experimental Determination of Cooperativity teins because their folding intermediates are stable The delineation of multiple steps, let alone gradual enough to accumulate and be easily detectable.8,11–34 transitions, is experimentally challenging. “Two- In such cases, structural characterization of inter- state” folding transitions have been proposed for mediates provides a wealth of information on folding many proteins, on the basis of studies that utilized pathways, and allows different folding pathways to just a single experimental probe, which can, howev- be distinguished from each other.29,35,36 A common er, be misleading.72 The hidden complexity of an class of intermediates which provide evidence for apparently simple “two-state” transition is often the reduced cooperativity of protein folding/unfold- revealed only by the use of multiple experimental ing reactions is that of the molten globular (MG) probes40,73,74 as well as different experimental condi- forms. The detection of dry molten globules,37–42 as tions.9,10 This is exemplified by studies on the pro- well as wet molten globules,43 demonstrates that tein monellin, using multiple methodologies (Fig. 1). changes in secondary and tertiary interactions, as Significant dispersion in the folding/unfolding rates well as changes in the solvation of the protein core, of different parts of a protein structure, demonstrat- may occur in multiple stages.19,44–47 When folding ed by monitoring multiple sites in the protein, have occurs in multiple steps via productive intermedi- provided direct evidence for asynchronous structural ates, the activation free energy to folding gets dis- changes which occur in multiple steps for many pro- tributed between multiple barriers. This results in teins.75–81 Several advances in the experimental faster folding than when the activation free energy techniques used to probe folding cooperativity82–86 is concentrated in one barrier in “two-state” folding. have yielded a quantitative understanding of ther- But intermediates could also be transient structures modynamic cooperativity, which pertains to the trapped in local energy minima because they possess number of states populated during a transition, as non-native interactions that have to be broken well as kinetic cooperativity, which pertains to the before folding can proceed.48,49 number of steps and free energy barriers between The dynamical coupling between the interac- the initial and final states of a reaction. tions which stabilize a well-packed N state deter- mines the cooperativity of the protein folding or Probing thermodynamic cooperativity unfolding reaction. Strong coupling between stabiliz- The classical way of determining the thermodynamic ing interactions would lead to an entirely coopera- cooperativity of a folding reaction is to determine tive two-state transition while a complete lack of the ratio of the calorimetric enthalpy to the van’t coupling would result in a gradual/continuous nonco- Hoff enthalpy, which is expected to be 1 for a two- operative transition. Gradual folding45,50–53 as well state process.87,88 A spectroscopic isobestic/isoemis- as unfolding54–62 structural transitions have now sive/isodichroic point will always be observed for been shown for several proteins. In fact, it has even equilibrium two-state unfolding, but it is not always been demonstrated that individual amino acid resi- remembered that the observation of such a single dues in a protein may have different equilibrium point where all the spectra for different stages of the unfolding transitions,63–68 providing strong evidence transition superimpose, need not necessarily mean for non-cooperativity in protein folding reactions. that the transition is two-state. Similarly, while mul- Barrier-limited two-state transitions, and barrier- tiple probes should yield coincident transition curves less gradual transitions are at opposite ends of the for two-state unfolding, the observation of coincident cooperativity spectrum, with the former being transition curves need not necessarily imply two- entirely cooperative, and the latter being entirely state unfolding. noncooperative. Multi-step transitions with discrete Analysis of equilibrium unfolding transitions, intermediates illustrate reduced/limited cooperativ- induced by denaturants or temperature, can yield ity in protein folding reactions: individual steps may other useful insights into the cooperative nature of be fully cooperative but the overall reaction is the unfolding reaction. For a true two-state process, noncooperative. the N and U protein baselines are expected to be Malhotra and Udgaonkar PROTEIN SCIENCE VOL 25:1924—1941 1925 Figure 1. Experimental determination of cooperativity. A: Single exponential kinetics of the unfolding of monellin probed by fluorescence (upper panel; kinetic traces of unfolding in increasing denaturant concentration are shown from right to left) sug- gested a “two-state” cooperative transition. B: Site-specific unfolding/opening of four cysteine side chains (upper panel), probed by thiol labeling (SX) under
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