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...

Orthogonal Peptide‐Templated Labeling Elucidates Lateral ETAR/ETBR Proximity and Reveals Altered Downstream Signaling

“…No cross reactivity of the two different coiled‐coil pairs was observed in our labeling experiments, which is consistent with the in depth characterization the P1/P2 and P3/4 coiled‐coils by Gradišar and Jerala [28] . Using this system, orthogonal labeling has been recently described for live cell imaging [61] . We employed peptide‐templated labeling for proximity‐dependent FRET analyses of four different GPCRs for which we detected varying FRET signals.…”

“… [28] Using this system, orthogonal labeling has been recently described for live cell imaging. [61] We employed peptide‐templated labeling for proximity‐dependent FRET analyses of four different GPCRs for which we detected varying FRET signals. High FRET signals point towards a close proximity of the GPCRs.…”

Synthesis of Protein-Oligonucleotide Conjugates

“…While disulfides are easily accessible from a synthetic standpoint, their reversibility must also be considered, especially in the harsh redox environment of a biological system. Finally, N-terminal cysteine residues undergo native chemical ligation (NCL) in the presence of an oligonucleotide bearing a C-terminal thioester ( Figure 2 D) [ 3 , 4 , 35 ]. An initial trans-thioesterification step links the two components, which is followed by a rapid S-to-N acyl shift to generate the stable amide bond linkage and regenerate the cysteine side chain.…”

“…The simplest way this can be achieved is through the use of bifunctional linkers, harnessing a pair of orthogonal chemical transformation [ 26 , 31 , 34 , 49 ]. Conversely, individual chemical conjugation techniques can be coupled with a templating element (ligand, protein–protein interaction, antibody, metal chelation) [ 3 , 4 , 22 , 27 , 32 , 50 , 51 , 52 , 53 ] which can be used to direct the conjugation to a specific region on a target protein, thereby improving the selectivity of the process ( Figure 3 ). This strategy relies on the recognition element (red) which binds to the target and displays an oligonucleotide strand.…”

“…For these reasons nucleic acids are often used for specific recognition of partner strands enabling the formation of defined nano objects, or for the physical transfer of information through conformational change by acting as molecular circuitry. It is important to note that such systems can rely equally on native DNA or on designer nucleic acids (such as peptide nucleic acids (PNA) [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ] or threoninol nucleic acids [ 11 ]) to fulfill this role. In contrast, proteins have a much wider array of potentially accessible structures as a result of their larger number of component building blocks, as well as the broader range of interactions these can undergo to influence global structure [ 1 ].…”