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Orthogonal coiled coils enable rapid covalent labelling of two distinct membrane proteins with peptide nucleic acid barcodes.

ABSTRACT: Templated chemistry offers the prospect of addressing specificity challenges occurring in bioconjugation reactions. Here, we show two peptide-templated amide-bond forming reactions that enable the concurrent labelling of two different membrane proteins with two different peptide nucleic acid (PNA) barcodes. The reaction system is based on the mutually selective coiled coil interaction between two thioester-linked PNA-peptide conjugates and two cysteine peptides serving as genetically encoded peptide tags. Orthogonal coiled coil templated covalent labelling is highly specific, quantitative and proceeds within a minute. To demonstrate the usefulness, we evaluated receptor internalisation of two membranous receptors EGFR (epidermal growth factor) and ErbB2 (epidermal growth factor receptor 2) by first staining PNA-tagged proteins with fluorophore-DNA conjugates and then erasing signals from non-internalized receptors via toehold-mediated strand displacement.

SUBMITTER: Gavins GC

PROVIDER: S-EPMC8341593 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Orthogonal coiled coils enable rapid covalent labelling of two distinct membrane proteins with peptide nucleic acid barcodes.

Gavins Georgina C GC Gröger Katharina K Reimann Marc M Bartoschek Michael D MD Bultmann Sebastian S Seitz Oliver O

RSC chemical biology 20210716 4

Templated chemistry offers the prospect of addressing specificity challenges occurring in bioconjugation reactions. Here, we show two peptide-templated amide-bond forming reactions that enable the concurrent labelling of two different membrane proteins with two different peptide nucleic acid (<b>PNA</b>) barcodes. The reaction system is based on the mutually selective coiled coil interaction between two thioester-linked <b>PNA</b>-peptide conjugates and two cysteine peptides serving as genetical ...[more]

PMID: 34458843

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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

Orthogonal coiled coils enable rapid covalent labelling of two distinct membrane proteins with peptide nucleic acid barcodes.

Publications

Orthogonal coiled coils enable rapid covalent labelling of two distinct membrane proteins with peptide nucleic acid barcodes.

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