The equilibrium unfolding pathway of a (β/α)8 barrel

Silverman, Julian; Harbury, Pehr B.

doi:10.1016/s0022-2836(02)01100-2

Cited by 65 publications

(100 citation statements)

References 30 publications

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“…Stable on-pathway intermediates built from cooperative foldon elements of the native protein are seen even under two-state folding conditions. 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: 85%

“…Thermodynamic principle requires that proteins repeatedly unfold and refold even under native conditions, revisiting their normal folding intermediates and recapitulating their normal folding process. All or parts of this process have been observed by site-resolved hydrogen exchange (19,(29)(30)(31)(32)(33)(34)(35), by a related thiol reactivity method (36), by NMR relaxation dispersion (37)(38)(39), and by theoretical analysis (40). Stable on-pathway intermediates built from cooperative foldon elements of the native protein are seen even under two-state folding conditions.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

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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“…We reasoned that cysteine residues placed within the DRS of ERK2 would serve to report the engagement of ligands at this locus through a decrease in their observed rate of alkylation. A related approach has been used to predict the TIP47 protein binding site for the mannose 6-phosphate receptor (30), as well as the substrate binding site of TIM (34). While ligand binding has the potential to increase or decrease k 1 /k −1 , and hence affect the reactivity of an ERK2-CT, by inducing a conformational change of a side chain or backbone segment, the relative inflexibility of the DRS suggests that for cysteines within the DRS a ligand-induced change in reactivity is likely to be the direct consequence of binding and not a conformational change (see the discussion below).…”

Section: Resultsmentioning

confidence: 99%

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

et al. 2007

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Many substrates of ERK2 contain a D-site, a sequence recognized by ERK2 that is used to promote catalysis. Despite lacking a canonical D-site, the substrate Ets-1 is displaced from ERK2 by peptides containing one. This suggests that Ets-1 may contain a novel or cryptic D-site. To investigate this possibility a protein footprinting strategy was developed to elucidate ERK2-ligand interactions. Using this approach, single cysteine reporters were placed in the D-recruitment site (DRS) of ERK2 and the resulting ERK2 proteins subjected to alkylation by iodoacetamide. The ability of residues 1-138 of Ets-1 to protect the cysteines from alkylation was determined. The pattern of protection observed is consistent with Ets-1 occupying a hydrophobic binding site within the DRS of ERK2. Significantly, a peptide derived from the D-site of Elk-1, which is known to bind the DRS, exhibits a similar pattern of cysteine protection. This analysis expands the repertoire of the DRS on ERK2 and suggests that other targeting sequences remain to be identified. Furthermore, cysteinefootprinting is presented as a useful way to interrogate protein-ligand interactions at the resolution of a single amino acid.The mitogen-activated protein kinases (MAPKs) 1 are ubiquitous elements of eukaryotic signaling pathways that permeate nearly all aspects of signaling in multicellular organisms. In humans the loss of their regulation is associated with many diseases that encompass an expanding list of cancers as well as neurological and inflammatory diseases (1). Three main subgroups have been identified in humans, termed the ERKs (2), the JNKs (3,4), and the p38 MAPKs (5,6). Several newer members have recently been reviewed (7). Extracellular regulated protein kinase 2 (ERK2), the prototypical member of the MAP kinase family, catalyzes the † This research was supported in part by the Welch Foundation (F-1390) and the National Institutes of Health (GM59802). Mass spectra were acquired by Dr. Herng-Hsiang Lo in the CRED Analytical Instrumentation Facility Core supported by the NIEHS center grant ES07784. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 July 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript transfer of the γ-phosphate of adenosine triphosphate to serine or threonine residues that generally lie in flexible regions of proteins. It is a remarkable enzyme whose ability to exhibit high specificity toward a discrete subset of structurally diverse proteins is poorly understood.Protein kinases generally recognize substrates through a consensus sequence that contains the phosphorylation site (8). This sequence is recognized, in part, by a region called the activation segment that encompasses residues 165 DFG to APE 195 of the catalytic domain of protein ki...

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“…The unfolding and refolding of cooperative foldon units and their role in forming intermediates and pathways has been detected, in greater or lesser detail, in many proteins by HX methods (23-25, 29, 47-55), by sulfhydryl labeling (56,57), by relaxation dispersion NMR (58), and in theoretical simulations (36)(37)(38). The inherent cooperativity of native-like foldon units predisposes them rather than other fractional elements to account for the unit steps in protein folding and thus dictates the stepwise formation and native-like nature of pathway intermediates.…”

Section: Discussionmentioning

confidence: 99%

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.

164

217

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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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The equilibrium unfolding pathway of a (β/α)8 barrel

Cited by 65 publications

References 30 publications

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

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

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

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

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