Folding and Unfolding Mechanism of Highly Stable Full-Consensus Ankyrin Repeat Proteins

Wetzel, Svava K.; Settanni, Giovanni; Kenig, Manca; Binz, Hans; Plückthun, Andreas

doi:10.1016/j.jmb.2007.11.046

Cited by 165 publications

(239 citation statements)

References 34 publications

(74 reference statements)

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“…Thereby, H 22 forms a hydrogen bond with the backbone of A 24 and is involved in the interaction network of the hydrophobic core by forming contacts with residue L 19 , N 24 and V 27 of the C-cap and E 25 and I 28 of the internal repeat ( Figure 9B). Notably, the same loop conformation is observed between internal repeats, although H 22 is replaced by D 22 there. In addition, the C PAF capping repeat has a nearly identical structure to the consensus-based A II -capping repeat [10] (PDB ID: 4DB6) ( Figure 9C).…”

Section: Computationally Designed Armadillo Scaffoldmentioning

confidence: 62%

“…This was achieved by exchanging exposed hydrophobic residues in the capping repeats to hydrophilic ones (N-cap: A 12 E, P 15 Q, L 19 W, V 27 T and A 34 Q; C-cap: V 8 E, V 17 E, L 20 Q, A 32 Q, A 36 E and A 39 N), as had been done for designed ankyrin repeat proteins [22] and designed ArmRPs from the consensus-design series [10].…”

Section: In Silico Designmentioning

confidence: 99%

“…for DARPins [22] and consensus-based ArmRPs [9,10]. For dArmRPs, the impact of the Y III -capping repeat was additionally tested on the original, computationally designed protein These results do not allow us to decide whether the capping repeat has an influence on the overall curvature or not.…”

Section: Stabilization Effect Of the Y III -Capping Repeat With The Dmentioning

confidence: 99%

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Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

Reichen

Hansen

Forzani

et al. 2016

Journal of Molecular Biology

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Armadillo repeat proteins (ArmRP) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. They may thus form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library.Stacked armadillo repeats, each consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed curvature variations in natural ArmRPs, and identified a repeat pair from yeast importin-α as having the optimal curvature geometry to be complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry.Computationally designed armadillo repeat proteins (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics (MD) simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series.

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Section: Computationally Designed Armadillo Scaffoldmentioning

confidence: 62%

Section: In Silico Designmentioning

confidence: 99%

Section: Stabilization Effect Of the Y III -Capping Repeat With The Dmentioning

confidence: 99%

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Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

Reichen

Hansen

Forzani

et al. 2016

Journal of Molecular Biology

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“…This combination of unstable repeats interacting through stabilizing interfaces is consistent with a recent Ising analysis of consensus ankyrin repeats. 19,21 Although the free energy landscape of the Notch ankyrin domain was determined from equilibrium unfolding measurements, several features of its shape may be relevant to the kinetics of folding. First, the landscape indicates high free energies for conformations that have only a few repeats folded, decreasing monotonically toward the native state, suggesting an early barrier in folding that makes a dominant contribution to folding kinetics.…”

Section: Resultsmentioning

confidence: 99%

“…Comparing folding free energies of repeat protein constructs with variable numbers of repeats can provide estimates for the intrinsic free energy of individual repeats and the interfacial free energy shared between neighboring repeats. 14,15,19 These intrinsic and interfacial free energies can be used to determine the free energies of partly folded states, including those too high in energy to be observed directly. Previous studies of the Notch ankyrin domain (see Fig.…”

Section: Introductionmentioning

confidence: 99%

Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

Street

Barrick

2008

Protein Science

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The Notch ankyrin domain is a repeat protein whose folding has been characterized through equilibrium and kinetic measurements. In previous work, equilibrium folding free energies of truncated constructs were used to generate an experimentally determined folding energy landscape (Mello and Barrick, Proc Natl Acad Sci USA 2004;101:14102-14107). Here, this folding energy landscape is used to parameterize a kinetic model in which local transition probabilities between partly folded states are based on energy values from the landscape. The landscape-based model correctly predicts highly diverse experimentally determined folding kinetics of the Notch ankyrin domain and sequence variants. These predictions include monophasic folding and biphasic unfolding, curvature in the unfolding limb of the chevron plot, population of a transient unfolding intermediate, relative folding rates of 19 variants spanning three orders of magnitude, and a change in the folding pathway that results from C-terminal stabilization. These findings indicate that the folding pathway(s) of the Notch ankyrin domain are thermodynamically selected: the primary determinants of kinetic behavior can be simply deduced from the local stability of individual repeats.

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Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development

Mehta

Cochran

2017

Methods and Principles in Medicinal Chemistry

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Folding and Unfolding Mechanism of Highly Stable Full-Consensus Ankyrin Repeat Proteins

Cited by 165 publications

References 34 publications

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development

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