Stabilisation of α-helices by site-directed mutagenesis reveals the importance of secondary structure in the transition state for acylphosphatase folding

Taddei, Niccolò; Chiti, Fabrizio; Fiaschi, Tania; Bucciantini, Monica; Capanni, Cristina; Stefani, Massimo; Serrano, Luís; Dobson, Christopher M.; Ramponi, Giampietro

doi:10.1006/jmbi.2000.3870

Cited by 54 publications

(38 citation statements)

References 58 publications

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“…Common-type acylphosphatase was expressed from a synthetic gene cloned into the pRSET B plasmid (Invitrogen) and purified as described in ref. 18. A 66-residue, amino-terminally cystine-linked coiled coil derived from the leucine zipper region of the protein GCN4 was synthesized and purified by analogy to Choma et al (19).…”

Section: Methodsmentioning

confidence: 99%

Random-coil behavior and the dimensions of chemically unfolded proteins

Kohn

Millett

Jacob

et al. 2004

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

657

758

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Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (RG) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The RG of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r2 = 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble

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Section: Methodsmentioning

confidence: 99%

Random-coil behavior and the dimensions of chemically unfolded proteins

Kohn

Millett

Jacob

et al. 2004

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

657

758

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“…This diffusion-collision model (D-C model) (10)(11)(12) is supported by the observation that helix formation is faster than overall folding rates (13)(14)(15). This broadly accepted view also is supported by the presence of helix in the folding transition state and an increase in k f with an increase in helical propensity (2,4,(16)(17)(18)(19)(20)(21).…”

mentioning

confidence: 91%

Fast folding of a helical protein initiated by the collision of unstructured chains

Meisner

Sosnick

2004

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

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To examine whether helix formation necessarily precedes chain collision, we have measured the folding of a fully helical coiled coil that has been specially engineered to have negligible intrinsic helical propensity but high overall stability. The folding rate approaches the diffusion-limited value and is much faster than possible if folding is contingent on precollision helix formation. Therefore, the collision of two unstructured chains is the initial step of the dominant kinetic pathway, whereas helicity exerts its influence only at a later step. Folding from an unstructured encounter complex may be efficient and robust, which has implications for any biological process that couples folding to binding.ne of the most debated issues in protein folding concerns the earliest folding events leading up to the transition state (1-9). For helical proteins, the earliest productive folding steps often are postulated to involve the collision of two preformed, but not necessarily stable, helical elements, rather than collision of unstructured chains. This diffusion-collision model (D-C model) (10-12) is supported by the observation that helix formation is faster than overall folding rates (13-15). This broadly accepted view also is supported by the presence of helix in the folding transition state and an increase in k f with an increase in helical propensity (2, 4, 16-21).However, this correlation can support an opposing model in which unstructured chains first collide, and the enhanced helicity increases the success frequency (or transmission coefficient) of each encounter (Fig. 1). In general, the highly cooperative (two-state) folding behavior of most small proteins precludes identifying the order of events leading up to the kinetic barrier. As a result, the demonstration of helical structure in the transition state cannot by itself resolve whether helix formation or chain collision occurs first.This obstacle can be overcome by studying a system with minimal helical propensity and composed of more than one chain, so that the rate of collision can be varied (22). These properties enable comparisons between observed folding rates and the maximum rate consistent with a model where precollision helix formation is required. Our investigation uses this strategy in conjunction with a dimeric coiled coil protein specially engineered to have negligible intrinsic helicity but high stability. This protein folds at nearly the diffusion limit and certainly at a much faster rate than would be possible if the helix must form before collision. Thus, an unstructured encounter complex can successfully initiate rapid folding, with helix formation occurring at a later step. The collision-first route sets a high basal level for the folding rate of any protein.

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“…Such conformational stabilization can be largely determined by an increase of folding rate, when the mutated helix is formed to a considerable extent in the folding transition state (5-7). Formation of local secondary structure to an extent similar to that existing in the transition state has been estimated to contribute to a significant, although small, fraction of the activation free energy required for folding (7). Recent experiments on SH3 domains from drkN and spectrin have shown that these ␤-sheet proteins can be strongly stabilized by single mutations that enhance the propensity of a region of the polypeptide chain to form the native ␤-turn structure (8,9).…”

mentioning

confidence: 99%

“…Two-state folding of AcP occurs through a transition state structure containing a primitive, not completely stabilized, hydrophobic core and significantly formed elements of secondary structure (16 -18). A study on the common-type AcP isoenzyme has shown that the second ␣-helix of the protein is completely formed in the transition state whereas the first is only partially structured (7). By means of an experimental approach conceptually similar to the value analysis introduced by Alan Fersht (19) for the structural characterization of the transition state of folding at a residue level, the aggregation rate has been investigated for a number of AcP mutants 2 .…”

mentioning

confidence: 99%

Folding and Aggregation Are Selectively Influenced by the Conformational Preferences of the α-Helices of Muscle Acylphosphatase

Taddei¹,

Capanni²,

Chiti³

et al. 2001

Journal of Biological Chemistry

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The native state of human muscle acylphosphatase (AcP) presents two ␣-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form ␤-sheet structure. The fact that the first ␣-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic ␣-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.

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Stabilisation of α-helices by site-directed mutagenesis reveals the importance of secondary structure in the transition state for acylphosphatase folding

Cited by 54 publications

References 58 publications

Random-coil behavior and the dimensions of chemically unfolded proteins

Random-coil behavior and the dimensions of chemically unfolded proteins

Fast folding of a helical protein initiated by the collision of unstructured chains

Folding and Aggregation Are Selectively Influenced by the Conformational Preferences of the α-Helices of Muscle Acylphosphatase

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