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Cooperative Folding Kinetics of BBL Protein and Peripheral Subunit-Binding Domain Homologues

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Cooperative Folding Kinetics of BBL Protein and Peripheral Subunit-Binding Domain Homologues Cooperative folding kinetics of BBL protein and peripheral subunit-binding domain homologues Wookyung Yu*, Kwanghoon Chung*†, Mookyung Cheon*‡, Muyoung Heo*§, Kyou-Hoon Han¶, Sihyun Hamʈ, and Iksoo Chang*,** *National Research Laboratory for Computational Proteomics and Biophysics, Department of Physics, Pusan National University, Busan 609-735, Korea; ‡Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom; ¶Molecular Cancer Research Center, Korea Research Institute for Bioscience and Biotechnology, Daejeon 305-806, Korea; and ʈDepartment of Chemistry, Sookmyoung Women’s University, Seoul 140-742, Korea Edited by Alan R. Fersht, University of Cambridge, Cambridge, United Kingdom, and approved December 21, 2007 (received for review September 7, 2007) Recent experiments claiming that Naf-BBL protein follows a global energy landscape when the folding energy predominates over the downhill folding raised an important controversy as to the folding loss of the conformational entropy (4), depending upon the mechanism of fast-folding proteins. Under the global downhill conditions of a folding experiment such as their initial locations folding scenario, not only do proteins undergo a gradual folding, on the free energy landscape and the heterogeneity of confor- but folding events along the continuous folding pathway also mational ensemble (7). In the global downhill folding model the could be mapped out from the equilibrium denaturation experi- folding free energy landscape possesses a global unimodal ment. Based on the exact calculation using a free energy landscape, distribution for all denaturation conditions so that a protein relaxation eigenmodes from a master equation, and Monte Carlo follows a smooth and progressive conformational change, and a simulation of an extended Muñoz–Eaton model that incorporates continuous folding pathway can be observed simply by an multiscale-heterogeneous pairwise interactions between amino equilibrium denaturation experiment (11, 12). acids, here we show that the very nature of a two-state cooper- Muñoz and coworkers (13, 14) presented the experimental ative transition such as a bimodal distribution from an exact free observation of a broad thermal denaturation midpoint for energy landscape and biphasic relaxation kinetics manifest in the Naf-BBL protein, which is a truncated BBL peripheral subunit thermodynamics and folding–unfolding kinetics of BBL and pe- binding domain (PSBD) from Escherichia coli with a naphthyl- ripheral subunit-binding domain homologues. Our results provide alanine. They not only suggested that Naf-BBL follows the global an unequivocal resolution to the fundamental controversy related downhill folding scenario but also claimed that the same scenario to the global downhill folding scheme, whose applicability to other holds for other PSBD homologues (13). In contrast, Fersht and proteins should be critically reexamined. coworkers (15, 16) examined the structural, denaturation, and kinetic properties of PSBD homologues from E. coli [unlabeled protein folding mechanism ͉ global downhill folding ͉ wild-type BBL, Protein Data Bank (PDB) entry 1W4H], Bacillus ͉ protein thermodynamics relaxation kinetics of protein stearothermophilus (E3BD F166W, PDB entry 1W4E), and BIOPHYSICS Pyrobaculumaerophilum (POB Y166W L146A, PDB entry ur understanding of protein folding is primarily based on 1W4J) (17) and found that 1W4H, 1W4E, and 1W4J follow the Ointerpretation of the thermodynamics and kinetics from a typical two-state folding scheme. Whereas Naganathan et al. (18) simple protein folding model (1–10). The principle of minimal argued that the experimental data in ref. 15 support the global frustration and structural consistency captured by the Go៮ model downhill folding scenario, Fersht and coworkers (16) believe that allowed us to describe the complicated nature of protein folding the conclusion drawn by Muñoz and coworkers was obtained by at a coarse-grained level (5, 6). Bryngelson et al. (6, 7) posited an incorrect assumption and analysis of the data in refs. 13 and a phenomenological picture of free energy landscape of a 18. More recently, Sadqi et al. (19) presented an atom-by-atom protein-like heteropolymer and classified protein folding into analysis of NMR data to support the global downhill folding three scenarios depending on whether the free energy landscape scheme of Naf-BBL (13). However, Ferguson et al. (20) and is unistable or bistable and also whether a glass transition occurs. Zhou and Bai (21) argued back again that the new data Shakhnovich and Gutin (8) presented microscopic statistical- presented by Sadqi et al. (19) could well be described by a two- mechanical theories for heteropolymer model, whose conse- or three-state model. Such an ongoing fundamental controversy quences provided important insights into the thermodynamics, (13–16, 18–25) about the folding mechanism for Naf-BBL and kinetics, and nature of free energy landscape for protein folding. PSBD homologues would reshape our concept on protein fold- The conventional view of protein folding assumes a discrete ing. Faced with these conflicting and ambiguous assertions from two-state transition between a native state and a denatured experiment, we have attempted to resolve such a controversy by ensemble of protein conformations that are separated by a carrying out an exact analysis of thermodynamic and kinetic folding barrier (9). The other view termed as one-state (or global) downhill folding asserts that there is a continuous change Author contributions: I.C. designed research; W.Y., K.C., M.C., M.H., and I.C. performed of protein conformation during folding without distinct free research; K.-H.H., S.H., and I.C. contributed new reagents/analytic tools; W.Y., K.C., M.C., energy barrier (7, 10). The former could be understood as the M.H., K.-H.H., S.H., and I.C. analyzed data; and I.C. wrote the paper. first order (cooperative) phase transition whereas the latter as The authors declare no conflict of interest. the second order (noncooperative) phase transition between a This article is a PNAS Direct Submission. native and a denatured ensemble of protein conformation. An †Present address: Ministry of Science and Technology, Gwacheon 427-715, Korea. in-depth analysis for possible downhill folding was performed by §Present address: Department of Chemistry and Chemical Biology, Harvard University, 12 Abkevich et al. (10) where it was shown that the major deter- Oxford Street, Cambridge, MA 02138. minant for the cooperativity of protein folding is the relative **To whom correspondence should be addressed. E-mail: [email protected]. abundance of nonlocal versus local contacts between residues in ac.kr. the native structure of a protein, which also simultaneously This article contains supporting information online at www.pnas.org/cgi/content/full/ compete with the loss of conformational entropy. However, the 0708480105/DC1. folding process of two-state proteins may go downhill in the free © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708480105 PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2397–2402 0 Fig. 1. Exact free energy ⌬G in the unit of RTm (Upper) and equilibrium population ␯ (Lower) plotted as a function of M for T/Tm ϭ 0.9ϳ1.1 for 1BBL (a), 1W4H (b), 1W4E (c), and 1W4J (d) using our extended ME model. The native (denatured) well is located at the global minimum of the free energy landscape for T Ͻ Tm (T Ͼ Tm). As T crosses Tm, the discrete transition occurs across the free energy barrier of 2ϳ3 RTm for 1BBL (a) and 1W4J (d) and 4ϳ5 RTm for 1W4H (b) and 1W4E (c), respectively. Because the native state is invariant, the peaks in ␯0 corresponding to the native state do not move with temperature, whereas the denatured peaks move to lower values of M while maintaining the bimodal distribution. The bimodal nature of eigenvector elements governs the equilibrium population. properties for folding–unfolding of an unlabeled BBL (BBL geneous pairwise interactions. Fig. 1 Upper shows an exact free short, PDB entry 1BBL) with no naphthyl moiety and three energy landscape in the unit of RTm as a function of the fraction PSBD homologues. In addition, a Monte Carlo simulation was M of native residue for 1BBL, 1W4H, 1W4E, and 1W4J, where used to monitor out-of-equilibrium relaxation kinetics. R is a gas constant and Tm is a folding mid-temperature. Although the free energy landscape of the denatured state is Results and Discussion broad and populated with multiple wells having low energy Thermodynamics of BBL and PSBD. The aforementioned contro- barriers with heights less than or equal to RTm, a clear free versy can be resolved by determining whether the thermody- energy barrier on the order of 2ϳ3 RTm for 1BBL and 1W4J, and namic and kinetic properties during folding–unfolding of BBL 4ϳ5 RTm for 1W4H and 1W4E exists that separates the native short and three PSBD homologues exhibit a bimodal or a from the denatured state. In addition, a discernible discrete jump unimodal pattern. To achieve such a goal one must use a free is identified in the location of the global minimum of free energy energy landscape in an exact manner along a proper reaction as T crosses Tm, which is a signature of the first order phase coordinate. A coarse-grained Muñoz–Eaton (ME) model of a transition between the native and the denatured ensemble of protein folding has been highly useful in explaining folding protein conformation. The invariant position of the native well processes of many proteins (26, 27). However, this original ME with temperature indicates that the native state is distinct and model does not distinguish different amino acid types. Further- constant. When T/Tm ϽϽ 1(T/Tm ϾϾ 1), the pairwise-contact more, pairwise interactions between two amino acids within a folding energy predominates over the loss of the conformational cut-off distance are treated as attractive interactions in this entropy; thus, the folding barrier diminishes and the folding model.
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