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Kinetic studies on strand displacement in de novo designed parallel heterodimeric coiled coils.

ABSTRACT: Among the protein folding motifs, which are accessible by de novo design, the parallel heterodimeric coiled coil is most frequently used in bioinspired applications and chemical biology in general. This is due to the straightforward sequence-to-structure relationships, which it has in common with all coiled-coil motifs, and the heterospecificity, which allows control of association. Whereas much focus was laid on designing orthogonal coiled coils, systematic studies on controlling association, for instance by strand displacement, are rare. As a contribution to the design of dynamic coiled-coil-based systems, we studied the strand-displacement mechanism in obligate heterodimeric coiled coils to investigate the suitability of the dissociation constants (K_D) as parameters for the prediction of the outcome of strand-displacement reactions. We use two sets of heterodimeric coiled coils, the previously reported N-A _x B _y and the newly characterized C-A _x B _y . Both comprise K_D values in the ?M to sub-nM regime. Strand displacement is explored by CD titration and a FRET-based kinetic assay and is proved to be an equilibrium reaction with half-lifes from a few seconds up to minutes. We could fit the displacement data by a competitive binding model, giving rate constants and overall affinities of the underlying association and dissociation reactions. The overall affinities correlate well with the ratios of K_D values determined by CD-thermal denaturation experiments and, hence, support the dissociative mechanism of strand displacement in heterodimeric coiled coils. From the results of more than 100 different displacement reactions we are able to classify three categories of overall affinities, which allow for easy prediction of the equilibrium of strand displacement in two competing heterodimeric coiled coils.

SUBMITTER: Groth MC

PROVIDER: S-EPMC5944379 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Kinetic studies on strand displacement in <i>de novo</i> designed parallel heterodimeric coiled coils.

Groth Mike C MC Rink W Mathis WM Meyer Nils F NF Thomas Franziska F

Chemical science 20180417 18

Among the protein folding motifs, which are accessible by <i>de novo</i> design, the parallel heterodimeric coiled coil is most frequently used in bioinspired applications and chemical biology in general. This is due to the straightforward sequence-to-structure relationships, which it has in common with all coiled-coil motifs, and the heterospecificity, which allows control of association. Whereas much focus was laid on designing orthogonal coiled coils, systematic studies on controlling associa ...[more]

PMID: 29780562

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Project description:ARCIMBOLDO solves the phase problem by combining the location of small model fragments using Phaser with density modification and autotracing using SHELXE. Mainly helical structures constitute favourable cases, which can be solved using polyalanine helical fragments as search models. Nevertheless, the solution of coiled-coil structures is often complicated by their anisotropic diffraction and apparent translational noncrystallographic symmetry. Long, straight helices have internal translational symmetry and their alignment in preferential directions gives rise to systematic overlap of Patterson vectors. This situation has to be differentiated from the translational symmetry relating different monomers. ARCIMBOLDO_LITE has been run on single workstations on a test pool of 150 coiled-coil structures with 15-635 amino acids per asymmetric unit and with diffraction data resolutions of between 0.9 and 3.0?Å. The results have been used to identify and address specific issues when solving this class of structures using ARCIMBOLDO. Features from Phaser v.2.7 onwards are essential to correct anisotropy and produce translation solutions that will pass the packing filters. As the resolution becomes worse than 2.3?Å, the helix direction may be reversed in the placed fragments. Differentiation between true solutions and pseudo-solutions, in which helix fragments were correctly positioned but in a reverse orientation, was found to be problematic at resolutions worse than 2.3?Å. Therefore, after every new fragment-placement round, complete or sparse combinations of helices in alternative directions are generated and evaluated. The final solution is once again probed by helix reversal, refinement and extension. To conclude, density modification and SHELXE autotracing incorporating helical constraints is also exploited to extend the resolution limit in the case of coiled coils and to enhance the identification of correct solutions. This study resulted in a specialized mode within ARCIMBOLDO for the solution of coiled-coil structures, which overrides the resolution limit and can be invoked from the command line (keyword coiled_coil) or ARCIMBOLDO_LITE task interface in CCP4i.

Dataset Information

Kinetic studies on strand displacement in de novo designed parallel heterodimeric coiled coils.

Publications

Kinetic studies on strand displacement in <i>de novo</i> designed parallel heterodimeric coiled coils.

Similar Datasets

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