Extremely Fast Folding of a Very Stable Leucine Zipper with a Strengthened Hydrophobic Core and Lacking Electrostatic Interactions between Helices

Dürr, Eberhard; Jelesarov, Ilian; Bosshard, Hans Rudolf

doi:10.1021/bi981891e

Cited by 82 publications

(92 citation statements)

References 68 publications

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“…To offset the stability lost because of low helical propensity, nine glutamic acids are introduced in each chain at positions next to the hydrophobic interface (26). Below pH 4, the carboxylic side chains protonate and form stabilizing tertiary interactions across the dimer interface, ''glutamic staples,'' by either hydrogen bonds or hydrophobic interactions.…”

Section: Resultsmentioning

confidence: 99%

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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Section: Resultsmentioning

confidence: 99%

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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“…The relatively low degree of α helicity estimated for the isolated subunits (especially H) is expected, given the generally accepted model for formation of coiled coils, which involves two largely unfolded monomers coming together to form a folded (hetero)dimer. 20,21 Additional evidence that the resulting EH heterodimer is the physiologically relevant species comes from thermal denaturation experiments, which show that the EH complex exhibits cooperative unfolding behavior, a hallmark of native protein structure. On the other hand, the cooperative loss of CD signal observed for the isolated E subunit probably does not indicate the unfolding of a native structure as it occurs at a temperature of ∼ 25°C, which is significantly below the optimum growth temperature for T. acidophilum (59°C).…”

Section: Discussionmentioning

confidence: 99%

The Stator Complex of the A1A0-ATP Synthase—Structural Characterization of the E and H Subunits

Kish-Trier

Briere

Dunn

et al. 2008

Journal of Molecular Biology

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Archaeal ATP synthase (A-ATPase) is the functional homolog to the ATP synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar ATPase found in the endomembrane system of eukaryotes. We have cloned, overexpressed and characterized the stator-forming subunits E and H of the A-ATPase from the thermoacidophilic Archaeon, Thermoplasma acidophilum. Size exclusion chromatography, CD, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and NMR spectroscopic experiments indicate that both polypeptides have a tendency to form dimers and higher oligomers in solution. However, when expressed together or reconstituted, the two individual polypeptides interact with high affinity to form a stable heterodimer. Analyses by gel filtration chromatography and analytical ultracentrifugation show the heterodimer to have an elongated shape, and the preparation to be monodisperse. Thermal denaturation analyses by CD and differential scanning calorimetry revealed the more cooperative unfolding transitions of the heterodimer in comparison to those of the individual polypeptides. The data are consistent with the EH heterodimer forming the peripheral stalk(s) in the A-ATPase in a fashion analogous to that of the related vacuolar ATPase.

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“…The relative importance of secondary versus tertiary interactions in determining folding rates was studied experimentally for a number of proteins, including the activation domain of carboxypeptyidase A (CPA) (84), CheY (84), the helical coiled-coil GCN4 (32,82,85,116), GB1 domain (19), its structural homologue protein L (57a), λ-repressor (12), and ribosomal protein L9 (73). In all cases, secondary interactions play no role or only a minor one in determining folding kinetics.…”

Section: Diffusion-collision Mechanismmentioning

confidence: 99%

Protein Folding Theory: From Lattice to All-Atom Models

Mirny

Shakhnovich

2001

Annu. Rev. Biophys. Biomol. Struct.

333

259

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This review focuses on recent advances in understanding protein folding kinetics in the context of nucleation theory. We present basic concepts such as nucleation, folding nucleus, and transition state ensemble and then discuss recent advances and challenges in theoretical understanding of several key aspects of protein folding kinetics. We cover recent topology-based approaches as well as evolutionary studies and molecular dynamics approaches to determine protein folding nucleus and analyze other aspects of folding kinetics. Finally, we briefly discuss successful all-atom Monte-Carlo simulations of protein folding and conclude with a brief outlook for the future.

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Extremely Fast Folding of a Very Stable Leucine Zipper with a Strengthened Hydrophobic Core and Lacking Electrostatic Interactions between Helices

Cited by 82 publications

References 68 publications

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

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

The Stator Complex of the A1A0-ATP Synthase—Structural Characterization of the E and H Subunits

Protein Folding Theory: From Lattice to All-Atom Models

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