Cytochrome c folds through foldon-dependent native-like intermediates in an ordered pathway Wenbing Hua,1, Zhong-Yuan Kana, Leland Maynea, and S. Walter Englandera,2 aPerelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Contributed by S. Walter Englander, February 5, 2016 (sent for review November 24, 2015; reviewed by Robert L. Baldwin and Doug Barrick) Previous hydrogen exchange (HX) studies of the spontaneous revers- folded native-like foldons will assist the subsequent formation of ible unfolding of Cytochrome c (Cyt c) under native conditions have adjacent foldons by way of native-like interactions [sequential led to the following conclusions. Native Cyt c (104 residues) is com- stabilization (11, 12)]. These properties will promote an ordered, posed of five cooperative folding units, called foldons. The high- stepwise, macroscopic folding pathway. energy landscape is dominated by an energy ladder of partially folded Fig. 1 compares the foldon-dependent defined pathway model forms that differ from each other by one cooperative foldon unit. The (10–13) and the undirected multipath energy landscape model reversible equilibrium unfolding of native Cyt c steps up through these (5, 14–16). The different models differ strikingly in the very dif- intermediate forms to the unfolded state in an energy-ordered se- ferent folding intermediates and pathways that they predict and quence, one foldon unit at a time. To more directly study Cyt c inter- might be distinguished thereby, but this effort has been difficult. mediates and pathways during normal energetically downhill kinetic One is asked to discern the structures of a whole set of kinetic folding, the present work used HX pulse labeling analyzed by a frag- intermediates that are neither fully unfolded (U) nor fully native – ment separation mass spectrometry method. The results show that (N), that cannot be isolated for study, and that only live for milli- 95% or more of the Cyt c population folds by stepping down through seconds. This task may seem impossible. Our earlier experiments the same set of foldon-dependent pathway intermediates as in ener- depended on an indirect approach, namely, investigation of the getically uphill equilibrium unfolding. These results add to growing dynamic reversible unfolding reactions that carry the native protein evidence that proteins fold through aclassicalpathwaysequenceof up through the high energy landscape. The results revealed that native-like intermediates rather than through a vast number of unde- cytochrome c (Cyt c) unfolds through a stepwise ladder of distinct finable intermediates and pathways. The present results also empha- – size the condition-dependent nature of kinetic barriers, which, with foldon-dependent intermediates (Fig. 1A, Right)(10 13). less informative experimental methods (fluorescence, etc.), are often The present work applied a hydrogen exchange (HX) pulse confused with variability in intermediates and pathways. labeling method that can provide a timed series of snapshots of the kinetic intermediates that are populated during normal ki- protein folding | foldons | cytochrome c netic folding, even when they form and dissipate on a time scale of milliseconds. Advanced HX–fragment separation–mass spec- trometry (HX MS) technology (17–20) can then read out the he protein-folding problem, one of the oldest and most dif- Tficult in all of biophysics, lies at the heart of biology. Proteins structure and progression of the transient protein folding inter- must fold to their active native state when they are initially re- mediates (21, 22). Unlike most previously used methods, which leased from the ribosome and when they unfold and refold, over present protein-folding signals in an unresolved whole-molecule and over again, during their lifetime (1). It is one reaction that and ensemble-averaged way (fluorescence, etc.), these methods all organisms—animals, plants, and microbes—absolutely re- separately specify folded and unfolded populations, resolve folding quire. However, 55 y after Anfinsen et al. showed that proteins events throughout the protein, and measure the time course of each can fold all by themselves without outside help (2), no generally one. The results show that Cyt c folds in a distinct foldon-dependent accepted folding model has emerged. kinetic pathway sequence. As suggested in Fig. 1A, the intermediates BIOPHYSICS AND Soon after Anfinsen’s demonstration, Levinthal noted that COMPUTATIONAL BIOLOGY sizeable proteins could not find their native state within any Significance reasonable folding time by a random search through the vast conformational space available to them (3). However, proteins The energy landscape model pictures that proteins fold through can fold in milliseconds. He concluded that proteins must fold many alternative pathways. The landscape contains all possible through some programmed structure formation pathway, analo- intermediates and pathways, and folding is driven only by the gous to other biochemical pathways, although one had no idea downhill energy gradient, with no way to choose any particular how such a pathway might look or what could guide it. A different folding step against the vast number of alternatives. On the answer to the Levinthal paradox, focused on energy landscape contrary, the present experiments demonstrate that Cytochrome considerations, points out that the search is not wholly random. c folds through distinct intermediates in a reproducible pathway, Refolding proteins must naturally diffuse energetically downhill adding one native-like foldon after another down a defined en- stepping through a landscape that contains all possible interme- ergy ladder. This behavior is dictated by the cooperative foldon diates and pathways. In the absence of more detailed direction, construction of the native protein. Two other proteins are known structural search processes at the microscopic, near single-residue to do the same. level must carry protein populations heterogeneously through all of these structural forms and different tracks, chosen perhaps by Author contributions: W.H., L.M., and S.W.E. designed research; W.H. and L.M. performed each protein’s initial unfolded conformation (4–7). research; W.H., Z.-Y.K., and L.M. analyzed data; and L.M. and S.W.E. wrote the paper. The discovery that proteins are constructed of cooperative Reviewers: R.L.B., Stanford University; and D.B., Johns Hopkins University. units called foldons (8–10) answers the Levinthal paradox in yet The authors declare no conflict of interest. another way. The foldon construction of native proteins provides Freely available online through the PNAS open access option. built-in instructions for assembling the native protein (10–12). 1Present address: Sorrento Pharmaceuticals, San Diego, CA 92121. Foldons are small enough, ∼20 residues, to be found quickly by 2To whom correspondence should be addressed. Email: [email protected]. random residue-level searching. Each foldon, being a cooperative This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. unit, will tend to fold in a stepwise, all-or-none manner. Already 1073/pnas.1522674113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1522674113 PNAS | April 5, 2016 | vol. 113 | no. 14 | 3809–3814 Downloaded by guest on September 28, 2021 ABTS G U search kcal/mol 12.8 10.0 C-helix 7.4 60’s-helix N-helix 6.0 4.3 0.0 N Fig. 1. Alternative folding models. (A) The defined pathway model previously inferred from HX studies of the foldon-dependent interconversion of native Cyt c with partially unfolded forms (PUFs) in the high free energy space above the native protein (10, 11, 13). (B) A representation of the energetically downhill multiple pathway model. Redrawn from ref. 16. These models are tested in the present work during kinetic folding. and the pathways that mediate uphill equilibrium unfolding and unfolding of all the foldons including the one called blue, is at downhill kinetic folding are the same. 13 kcal/mol above N. It exists in the high free energy space above −10 N with unfolding equilibrium constant Kunf ∼ 10 . At [GdmCl] = Results 1.5 M, the blue unit unfolding reaction is promoted (ΔGunf ∼ Equilibrium Foldon Unfolding by NMR. Foldons were first defined in 6 kcal/mol above N) and comes to dominate the exposure and the −5 equilibrium native-state HX experiments with Cyt c (9), repro- HX of the amides in the blue foldon (Kunf ∼ 10 ). duced in Fig. 2A. HX rates of many individual amides were mea- These results point to four cooperative foldon unfolding reac- sured by 2D NMR as a function of increasing, but still very low, tions, some more clearly than others. Seven residues through the denaturant concentration, well below the Cyt c melting zone. Fig. N-terminal helix and eight through the C-terminal helix are seen 2A plots the HX rates measured for each residue amide in terms of to join into the same high free energy denaturant-sensitive HX the free energy of the opening reaction that exposes it to exchange. isotherm (blue). One infers that a concerted transient unfolding Amides that are exposed to exchange by small opening reactions reaction involves both terminal helices (blue). Similarly, five res- (local fluctuations) are insensitive to denaturant and trace hori- idues in the 60s helix and one in a nearby omega loop participate zontal curves. Larger unfolding reactions, otherwise invisible, are in a different concerted unfolding reaction with smaller unfolding sharply promoted