Protein folding: The stepwise assembly of foldon units

Maity, Haripada; Maity, Mita; Mallela, Krishna; Mayne, Leland; Englander, S. Walter

doi:10.1073/pnas.0501043102

Cited by 291 publications

(352 citation statements)

References 39 publications

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“…The folding model pictured in Fig. 5B is identical to the extensively worked out case of cytochrome c for which a series of HX NMR studies defined four native-like foldon-based intermediates in a well-ordered pathway (24)(25)(26). It also is consistent with the finding of individual nativelike intermediates in many other proteins.…”

Section: Discussionsupporting

confidence: 77%

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Hu¹,

Walters

Kan³

et al. 2013

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

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The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on nativelike interfoldon interactions orders the pathway.D o proteins fold through varied and multiple tracks, or do they fold through predetermined intermediates according to understandable biophysical principles (1)? This question is fundamental for the interpretation of a large amount of biophysical and biological research. The question could be resolved if it were possible to define the intermediate structures and pathways that unfolded proteins move through on their way to the native state. Unfortunately, transient intermediates cannot be studied by the usual crystallographic and NMR methods. The range of kinetic and spectroscopic methods has been applied to many proteins, but these methods do not yield the necessary structural information.We used a developing technology, hydrogen exchange pulse labeling measured by MS (HX MS), to study the folding of a cysteine-free variant of Escherichia coli ribonuclease H1 (RNase H), a mixed α/β protein that has served as a major proteinfolding model (2-5). Previous studies showed that RNase H folds in a fast, unresolved burst phase (15 ms dead time) to an intermediate termed "I core " and then much more slowly (in seconds) to the native state (3). HX pulse-labeling and equilibrium native-state HX experiments monitored by NMR showed that I core comprises a continuous region of the protein between helix A and strand 5 and that β-strands 1, 2, and 3 and helix E acquire protection much later, consistent with mutational analysis (2-4). Single-molecule and mutational studies indicated that the intermediate is obligatory, on-pathway, and folds first even when I core is not observably populated (6, 7).The HX MS technique used here is able to follow the entire folding trajectory of RNase H in considerable structural and temporal detail. The analysis monitors every amide site, evaluates the folding coopera...

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Section: Discussionsupporting

confidence: 77%

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Hu¹,

Walters

Kan³

et al. 2013

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

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165

217

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“…This has been shown for Cyt c (41), triose phosphate isomerase (36), RNase H (19), OspA (33), and apoCyt b 562 (30,32). Furthermore, detailed pathway information indicates that native-like foldon units are systematically put into place in well defined sequential pathways much as they fit together in the target native protein (19,30,36,(41)(42)(43)(44)(45)(46)(47)(48).…”

Section: Discussionmentioning

confidence: 86%

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

Bédard

Mallela

Mayne

et al. 2008

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

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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...

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“…These same foldon units have been repeatedly confirmed in multiple experiments under different conditions by HX pulse labeling (Krishna et al 2003a(Krishna et al , 2004b, by equilibrium NHX (Bai et al 1995b;Bai & Englander, 1996;Englander et al 1997;Krishna et al 2003bMaity et al 2005), and by a related kinetic NHX method that distinguishes concerted foldon units by virtue of their different unfolding rates rather than their different stabilities and m values (Hoang et al 2002;Krishna et al 2004a). …”

Section: 22mentioning

confidence: 79%

Protein folding and misfolding: mechanism and principles

Englander

Mayne

Mallela

2007

Quart. Rev. Biophys.

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267

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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

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Protein folding: The stepwise assembly of foldon units

Cited by 291 publications

References 39 publications

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

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

Protein folding and misfolding: mechanism and principles

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