Apparent two-state tendamistat folding is a sequential process along a defined route11Edited by A. R. Fersht

The 33-amino-acid ankyrin motif comprises a β-turn followed by two anti-parallel α-helices and a loop and tandem arrays of the motif pack in a linear fashion to produce elongated structures characterized by short-range interactions. In this article we use site-directed mutagenesis to investigate the kinetic unfolding mechanism of D34, a 426-residue, 12-ankyrin repeat fragment of the protein ankyrinR. The data are consistent with a model in which the N-terminal half of the protein unfolds first by unraveling progressively from the start of the polypeptide chain to form an intermediate; in the next step, the C-terminal half of the protein unfolds via two pathways whose transition states have either the early or the late C-terminal ankyrin repeats folded. We conclude that the two halves of the protein unfold by different mechanisms because the N-terminal moiety folds and unfolds in the context of a folded C-terminal moiety, which therefore acts as a “seed” and confers a unique directionality on the process, whereas the C-terminal moiety folds and unfolds in the context of an unfolded N-terminal moiety and therefore behaves like a single-domain ankyrin repeat protein, having a high degree of symmetry and consequently more than one unfolding pathway accessible to it

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confidence: 99%

Shifting transition states in the unfolding of a large ankyrin repeat protein

Werbeck

Rowling

Chellamuthu

et al. 2008

Section: Resultsmentioning

confidence: 99%

“…Equilibrium denaturation curves for both R16 and R17 mutants fitted well to a two-state transition (36). All R16 mutant chevron plots were fitted using the sequential transition state model (17,(19)(20)(21)(22)(23). In urea, the parameters (m 2 ¼ −0.7 M −1 and m −2 ¼ 0.5 M −1 ), obtained from global fitting of R16 wild-type and its large set of Φ-value mutants (17), were used to constrain the fits of the data presented herein.…”

Section: Methodsmentioning

confidence: 99%

“…The curvature is most pronounced for K25V but present for all mutants. A number of models have been used to fit such downward curvature; the model that has been shown to best fit R16, and its mutants, is a sequential transition state model with two obligatory, sequential transition states separated by a high-energy intermediate (17,(19)(20)(21)(22)(23).The most obvious difference between R16 and R16m5 (which contains all five of these substitutions) is the significant increase in both k f and k u . Three of the substitutions are fairly conservative (E19D, I22L, and V29L), and individually these have little effect on the folding or unfolding kinetics (with Φ-values estimated close to 0 or 1, from visual inspection of the chevron plots and calculation) ( Fig.…”

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confidence: 99%

See 1 more Smart Citation

Separating the effects of internal friction and transition state energy to explain the slow, frustrated folding of spectrin domains

Wensley

Kwa

Shammas

et al. 2012

The elongated three-helix bundle domains spectrin R16 and R17 fold some two to three orders of magnitude more slowly than their homologue R15. We have shown that this slow folding is due, at least in part, to roughness in the free-energy landscape of R16 and R17. We have proposed that this roughness is due to a frustrated search for the correct docking of partly preformed helices. However, this accounts for only a small part of the slowing of folding and unfolding. Five residues on the A helix of R15, when inserted together into R16 or R17, increase the folding rate constants, reduce landscape roughness, and alter the folding mechanism to one resembling R15. The effect of each of these mutations individually is investigated here. No one mutation causes the behavior seen for the five in combination. However, two mutations, E18F and K25V, significantly increase the folding and unfolding rates of both R16 and R17 but without a concomitant loss in landscape roughness. E18F has the greatest effect on the kinetics, and a Φ-value analysis of the C helix reveals that the folding mechanism is unchanged. For both E18F and K25V the removal of the charge and resultant transition state stabilization is the main origin of the faster folding. Consequently, the major cause of the unusually slow folding of R16 and R17 is the non-native burial of the two charged residues in the transition state. The slowing due to landscape roughness is only about fivefold.free energy landscape | frustration | phi value | protein folding T he 15th, 16th, and 17th domains of chicken brain α-spectrin (R15, R16, and R17) have very similar structures, stabilities, and β-Tanford (β T ) values (which reflect the compactness of the transition state for folding and unfolding) (1-6). However, the folding of R15 differs from that of R16 and R17 in a number of respects. R15 folds and unfolds two orders of magnitude faster than R16 and three orders of magnitude faster than R17. R16 and R17 have two sequential transition states, and for both domains the first of these (TS1) shows significant landscape roughness (or "internal friction") (7). This has not been seen for any other domain of comparable size and folding kinetics, although theory has long predicted the possibility of such landscape roughness (8-15). R15 has a broad transition state (characterized by "rollover" in the unfolding limb for some mutants and for wild type under some conditions), but due to the speed of folding and unfolding it is not known whether it has two sequential transition states (16). However, the early transition state of R15 (which corresponds to TS1 of R16 and R17 and will be referred to as such) has a smoother, less frustrated landscape (7).Φ-value analysis shows that for all three domains the A and C helices are partially structured at TS1, whereas the B helix is relatively unstructured (16-18). R16 and R17 fold via a framework-like mechanism, with some tertiary contacts formed but more extensive secondary structure that extends throughout the A and C helices. In contrast, R1...

“…Folding mechanism switches are not so unusual, nonetheless. For some polypeptides, curved chevron plots have been observed only in some variants (44,45), members of the protein family (46), or solvent conditions (47,48).…”

Section: Discussionmentioning

confidence: 99%

Hydrogen-exchange stability analysis of Bergerac -Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

Viguera

Serrano

2003

Amide hydrogen͞deuterium exchange rates have been determined for two mutants of ␣-spectrin Src homology 3 domain (WT), containing an elongated stable (SHH) and unstable (SHA) distal loop. SHA, similarly to WT, follows a two-state transition, whereas SHH apparently folds via a three-state mechanism. Native-state amide hydrogen exchange is effective in ascribing energetic readjustments observed in kinetic experiments to species stabilized within the denatured base and distinguishing those from highenergy barrier crossings. Comparison of ⌬G ex and mex parameters for amide protons of these mutants demonstrates the existence of an intermediate and allows the identification of protons protected in this state. The consolidation of a form containing a prefolded long ␤-hairpin induces the switch to a three-state mechanism in an otherwise two-state folder. It can be inferred that the unbalanced high stability of individual elements of secondary structure in a polypeptide could ultimately complicate its folding mechanism.kinetics ͉ ␤-hairpin T he Src homology 3 (SH3) domain from ␣-spectrin is a wellcharacterized model system for protein folding and stability (1). It was shown early that this domain folds by a two-state transition (2) and that its isolated secondary structure elements are quite unstructured (3). We have investigated (4) the consequences of fusing the SH3 domain with a short sequence KITVNGKTYE (BHH) that tends to fold as a stable ␤-hairpin (5). In the SHHBergerac mutant (SHH for short) the two-residue distal ␤-turn (N47-D48) of ␣-spectrin SH3 has been substituted by BHH. SHH is more stable and folds significantly faster than the short WT ␣-spectrin SH3 at moderate urea concentrations, but unlike WT, its folding rate levels off under strong folding conditions (Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org), thus deviating from the simplest two-state folding mechanism. Mutation of two residues in the inserted BHH region of SHH (Ile-2Ј and Val-4Ј to Ala, SHA-Bergerac) destabilizes the protein and switches it back to a two-state folding process (Fig. 6). When fused to WT, the inserted BHH and BHH I2ЈA V4ЈA sequences adopt a fully folded ␤-hairpin structure protruding from the protein (4), but the corresponding isolated peptides are, respectively, Ϸ30% folded (BHH) and fully unfolded (BHH I2ЈA V4ЈA).Understanding the reasons behind rate constant deviations could be important in improving the methods available for in vitro protein stabilization, as well as in revealing limiting steps related to switches from two to higher order folding mechanisms. Deviations in chevron plots do not have an unequivocal interpretation. Rate constants reflect free energy differences from reactants to the highest energy level in the reaction coordinate or transition state ( ‡). The deviations often occurring at lower denaturant concentrations in refolding experiments are usually assigned to changes within the denatured ensemble, i.e., a new energy level or intermediate state (I) is stabilized...