The folding energy landscape of apoflavodoxin is rugged: Hydrogen exchange reveals nonproductive misfolded intermediates

Proc. Natl. Acad. Sci. U.S.A.

et al. 2008

The observation of heterogeneous protein folding kinetics has been widely interpreted in terms of multiple independent unrelated pathways (IUP model), both experimentally and in theoretical calculations. However, direct structural information on folding intermediates and their properties now indicates that all of a protein population folds through essentially the same stepwise pathway, determined by cooperative native-like foldon units and the way that the foldons fit together in the native protein. It is essential to decide between these fundamentally different folding mechanisms. This article shows, contrary to previous supposition, that the heterogeneous folding kinetics observed for the staphylococcal nuclease protein (SNase) does not require alternative parallel pathways. SNase folding kinetics can be fit equally well by a single predetermined pathway that allows for optional misfolding errors, which are known to occur ubiquitously in protein folding. Structural, kinetic, and thermodynamic information for the folding intermediates and pathways of many proteins is consistent with the predetermined pathway-optional error (PPOE) model but contrary to the properties implied in IUP models.foldons ͉ PPOE model ͉ staphylococcal nuclease T hat proteins might fold by way of some predetermined pathway was first suggested by C. Levinthal (1), who reasoned that folding through a random search process would take far longer than the subsecond time scale often observed. Experimental work driven by this concept found evidence for distinct pathway intermediates (2). However, theoretical work showed that the energetically downhill nature of the conformational search might by itself be sufficient to drive random folding on a realistic time scale (3). No specific pathway would then be necessary. This scenario has been formalized in the funneled energy landscape view for protein folding (4-8). Following this precedent, experimental results for a number of proteins have been similarly interpreted in terms of multiple unrelated parallel pathways (9)(10)(11)(12)(13)(14)(15)(16). The critical observation is that protein folding can be kinetically heterogeneous. Different fractions of a folding population fold at different rates and exhibit different populated intermediates, suggesting alternative pathways. This view is often represented in terms of multiple tracks through the funneled energy landscape. We refer to this view as the independent unrelated pathways (IUP) model to emphasize that intermediate structures in the different tracks have no particular relationship.A much more determinate pathway is supported by structural information derived experimentally for folding intermediates in many proteins (17)(18)(19)(20). This information indicates that protein folding intermediates are definitively native-like, constructed from cooperative foldon units of the target native protein. It appears that native-like foldon units are formed and docked into place one after another in a stepwise sequence, each one guided and stabilized by pr...

Section: Discussionmentioning

confidence: 99%

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

Bédard

Mallela

Proc. Natl. Acad. Sci. U.S.A.

et al. 2008

“…In unfolded forms, some of the interactions that delimit structural elements in the native protein are absent and other non-native energy-minimizing interactions may be present. Accordingly, one often finds evidence in partially folded intermediates for non-native interactions in addition to strikingly native-like conformation (Radford et al 1992;Capaldi et al 2002;Krishna et al 2004b;Feng et al 2005a;Bollen et al 2006;Neudecker et al 2007). Most noteworthy, Bai and colleagues solved the NMR solution structures of two apoCyt b 562 PUFs (Feng et al 2003a(Feng et al , 2005a.…”

Section: Foldon Structurementioning

confidence: 99%

Protein folding and misfolding: mechanism and principles

Englander

Mallela

2007

Quart. Rev. Biophys.

172

267

Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding. The protein folding problemThe search for protein folding pathways and the principles that guide them has proven to be one of the most difficult problems in all of structural biology. Biochemical pathways have almost universally been solved by isolating the pathway intermediates and determining their structures. This approach fails for protein folding pathways. Folding intermediates only live for <1 s and they cannot be isolated and studied by the usual structural methods.The protein folding problem has been attacked by the entire range of fast spectroscopic methods which are able to track folding in real time. Much valuable information has been obtained but mainly of a kinetic nature. Kinetic methods do not describe the structure of the

“…Misfolding in partially unfolded forms has often been seen before. 16,22,[46][47][48][49] Residue 25 is exposed to HX with the rate and equilibrium parameters of the blue foldon even though it is placed across the β2-β3 hairpin in the yellow foldon. No other β2-β3 residue is protected in this way, not even residue 32 which is the paired partner of residue 25 in the native protein.…”

Section: Misfoldingmentioning

confidence: 99%

The Foldon Substructure of Staphylococcal Nuclease

Bédard

Journal of Molecular Biology

Peterson

et al. 2008

To search for submolecular protein foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease (SNase) under native conditions was studied by a kinetic native state hydrogen exchange (NHX) method. As for other proteins, it appears that SNase is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measures the equilibrium stability (ΔG HX ) and the kinetic unfolding and refolding rates (k op and k cl ) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with rate constant of 6x10 −6 s −1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel including mutually Hbonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of theα2 helix which is capped by β5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1-β2-β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although structure of the partially unfolded forms (PUFs) closely mimics the native organization, four residues indicate the presence of some non-native misfolding interactions. Because the unfolding parameters of many other residues are not determined it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.