ISSN 0006-2979, Biochemistry (Moscow), 2018, Vol. 83, Suppl. 1, pp. S33-S47. © Pleiades Publishing, Ltd., 2018. Original Russian Text © V. E. Bychkova, G. V. Semisotnov, V. A. Balobanov, A. V. Finkelstein, 2018, published in Uspekhi Biologicheskoi Khimii, 2018, Vol. 58, pp. 67-100. REVIEW The Molten Globule Concept: 45 Years Later V. E. Bychkova*, G. V. Semisotnov, V. A. Balobanov, and A. V. Finkelstein Institute of Protein Research, 142290 Pushchino, Moscow Region, Russia; E-mail: [email protected] Received June 15, 2017 Revision received July 18, 2017 Abstract—In this review, we describe traditional systems where the molten globule (MG) state has been detected and give a brief description of the solution of Levinthal’s paradox. We discuss new results obtained for MG-mediated folding of “non- traditional” proteins and a possible functional role of the MG. We also report new data on the MG, especially the dry molten globule. DOI: 10.1134/S0006297918140043 Keywords: protein self-organization folding, phase transitions in proteins, kinetics of protein folding, molten globule, for- mation and association of protein secondary structure, free-energy barrier, Levinthal’s paradox To the memory of Oleg B. Ptitsyn Forty-five years ago, Oleg B. Ptitsyn proposed a protein–protein interactions, etc. That review also cov- hypothesis of a stepwise mechanism of protein self- ered the equilibrium molten globule, phase transitions in organization [1]. The available data on renaturation of proteins, kinetics and mechanisms of protein folding, and globular proteins indicated that the primary structure of a the physiological role of the molten globule. protein carries information about its spatial organization The years after publication of this review have [2, 3]. However, the protein chain is simply unable to brought better understanding of how a polypeptide chain sample all possible conformations in search for its unique finds the right way to its native, functional structure, spatial structure during a reasonable time (this is the so- which intermediates and transitional states it must over- called “Levinthal’s paradox” [4]). Therefore, it was only come, and what is the energy of this process (see reviews natural to assume that protein self-organization is a [6-17] and references therein). Novel methods have been directed multi-step process (Fig. 1). With almost no developed and applied to protein folding studies; they experimental results to proceed from at the time (1973), have revealed various details of the process of protein Ptitsyn proposed that the key step in the process is the structure formation. In the same years, the attention of formation of what we now call the “molten globule” researchers was shifted towards protein “misfolding”, (MG) [1]. especially amyloid formation [6]. In 1995 Ptitsyn published his extensive, detailed, and most informative review on the “Molten Globule and Protein Folding” [5], where this intermediate was shown HISTORICAL EXCURSUS: THE MOLTEN to occur not only in the course of protein folding, but also GLOBULE IS A COMMON INTERMEDIATE during its denaturation, interaction with membranes, IN THE PATHWAY OF GLOBULAR PROTEIN SELF-ORGANIZATION Abbreviations: ANS, 8-anilino-1-naphthalenesulfonic acid; apoMb, apomyoglobin; ASA, accessible surface area; Bα-LA, This section of the current review relates to the 50th bovine alpha-lactalbumin; β-LG, β-lactoglobulin; DHFR, dihy- anniversary of the Institute of Protein Research, Russian drofolate reductase; FRET, fluorescence resonance energy trans- Academy of Sciences, and to the same anniversary of the fer; Hα-LA, human alpha-lactalbumin; HSQC, heteronuclear single-quantum correlation (of NMR spectra); MG, molten Protein Physics Laboratory that Ptitsyn founded at the globule; N, I, U are native, intermediate, unfolded state, respec- Institute. Here we focus on Ptitsyn’s concept (1973) that tively; TS, transition state; TTET, triplet–triplet energy transfer; self-organization of a protein molecule necessarily results WMG and DMG, “wet” and “dry” molten globule, respectively. from a directed process encoded in its amino acid * To whom correspondence should be addressed. sequence and realized in time as a series of steps. S33 S34 BYCHKOVA et al. Secondary Native-like by local interactions, whose strength and specificity is not structure secondary large [19, 20, 23, 25]. fluctuating structure and Native Secondary structure. The formation of secondary Unfolded around its folding motif of spatial structure is of great importance because it not only chain native position protein chain structure decreases the chain volume and helps its transition from the aqueous medium to the globule, but it also contributes to correct topology of the chain (its spatial architecture), which is already an element of the tertiary structure. Experiments in support of this concept were reported later ([27], see also [47, 75, 78-80]) showing that H-bonding Fig. 1. The stepwise model of protein folding [1]. Secondary struc- leads to protein compaction when a urea-unfolded chain ture elements are shown: α-helices (cylinders) and β-sequences is transferred into a “poor” solvent (water in this case) (arrows). Both predicted intermediates were later experimentally [27, 28]. At early stages of protein folding, seeds of sec- demonstrated and termed the “pre-molten” and “molten” glob- ondary structure that is afterwards observed in the native ules. protein usually appear. An interesting exception is exem- plified by beta-lactoglobulin (β-LG) [29-32]. It has an At that time, Ptitsyn’s team was developing a theory early kinetic intermediate with a nonnative α-helix whose of formation of the secondary structure of globular pro- presence there before the globule has been formed is not a teins [18-25]. They showed that the stabilization of heli- great surprise, because the β-LG site containing this helix cal and elongated β-structural regions in the protein is known to encode precisely an α-helical structure; fur- globule is determined by interactions between distant ther transition of this helix to β-structure is dictated by chain regions rather than local ones. However, local tertiary interactions. This example shows that the men- interactions alone are good enough for finding the sec- tioned coordination of different interactions in a protein ondary structure localization because they are related to has a statistical rather than absolute character [26]. long-range interactions that stabilize helical and β-struc- The MG, an equilibrium intermediate state. Ptitsyn’s tural regions exactly at the sites of local interactions hypothesis proposed in 1973 was developed in his subse- within the unfolded chain. (Later, it was shown that sta- quent works [21, 23, 33-35]. Concurrently, experimental tistically reliable matching of different interactions is studies of protein denaturation revealed an equilibrium necessary for the stability of a protein structure [26].) intermediate [36] termed the “molten globule” (MG) The further developed theories on other secondary struc- [37] that showed properties of the folding intermediate ture elements, such as bends and loops, showed that their predicted in 1973 (see [10, 34, 36], [38] and references localization also originates from local interactions and therein, [39]). Studies of protein folding kinetics also con- are then stabilized by long-range interactions during for- firmed the existence of kinetic intermediates of the MG mation of the globular structure [1, 18-25]. In other type (see [5] and references therein, [34, 35, 38, 40, 41]). words, the type and localization of protein secondary The Protein Physics Lab was broadly involved in structures are largely determined by the local amino acid these studies ([41-44], also see references in review [45]). sequence, although this structure is stabilized mostly by Specifically, a method using MG staining with ANS dye long-range rather than local interactions between chain was proposed to test the accessibility of the hydrophobic regions. MG core for solvents [43, 44, 46]. This method proved to Ptitsyn’s hypothesis implied that the fluctuating sec- be most useful in identifying the equilibrium MG [46]. ondary structure seeds originate mostly from local inter- As emphasized by Ptitsyn’s hypothesis, the forma- actions (primarily H-bonding) within the unfolded pro- tion of a native-like topology is a must for the existence of tein chain. Then they coalesce to form a compact globu- the MG. This requirement was later demonstrated for lar structure (Fig. 1), which is not yet the unique native bovine alpha-lactalbumin (Bα-LA) [47, 75, 78], a cold structure, but only an intermediate resulting mostly from shock protein [48], and other proteins [10, 49]. nonspecific interactions between amino acid residues and Proteins folding with intermediates (multi-state pro- the surrounding medium (water and the hydrophobic teins) and without intermediates (two-state proteins). core of the incipient globule) [1, 21]. Studies of protein folding kinetics revealed that proteins According to Ptitsyn’s hypothesis, further formation mostly fold via their intermediate states formed in the of the unique native structure consists of adjusting this folding pathway [41, 49, 50]. Proteins having more than intermediate, where the main role should be played by two states (native, one, or several intermediates, and fully specific forces of interaction between adjacent amino acid unfolded) are called “multi-state proteins”. In their fold- residues. This hypothesis