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Synthetic molecules that form a covalent bond upon binding to a targeted biomolecule (proximity-induced reactivity) are the subject of intense biomedical interest for the unique pharmacological properties imparted by irreversible binding. However, off-target covalent labeling and the lack of molecules with sufficient specificity limit more widespread applications. We describe the first example of a cross-linking platform that uses a synthetic peptide epitope and a single domain antibody (or nanobody) pair to form a covalent linkage rapidly and specifically. The rate of the cross-linking reaction between peptide and nanobody is faster than most other biocompatible cross-linking reactions, and it can be used to label live cells expressing receptor-nanobody fusions. The rapid kinetics of this system allowed us to probe the consequences on signaling for ligand cross-linking to the A2A-adenosine receptor. Our method may be generally useful to site-specifically link synthetic molecules to receptors on mammalian cell surfaces.

Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species. The latter approach necessitates the creation of fusions whose behavior may not accurately reflect that of their unmodified counterparts. Here, we report a method for the characterization of protein internalization in real time through sortase-mediated, site-specific labeling of single-domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. Quenched probes of this type have been used to measure enzyme activity in complex environments and for different cell types, but not as a sensor of protein movement into living cells. This approach allows a quantitative assessment of the movement of proteins into protease-containing endosomes in real time in living cells. We demonstrate considerable variation in the rate of endosomal delivery for different cell surface receptors. We were also able to characterize the kinetics of influenza virus delivery to cathepsin-positive compartments, showing highly coordinated arrival in endosomal compartments. This approach should be useful for identifying proteins expressed on cells of interest for targeted endosomal delivery of payloads, such as antibody-drug conjugates or antigens that require processing.

Oligomers that contain both α- and β-amino acid residues, or "α/β-peptides", have emerged as promising mimics of signal-bearing polypeptides that can inhibit or augment natural protein-protein interactions. α/β-Peptides that contain a sufficient proportion of β residues evenly distributed along the sequence can be highly resistant to enzymatic degradation, which is favorable with regard to in vivo applications. Little is known, however, about recognition of α/β-peptides by the immune system. Prior studies have focused almost entirely on examples that contain a single β residue; such α/β-peptides frequently retain the immunological profile of the analogous α-peptide. We have conducted α-peptide vs α/β-peptide comparisons involving higher β residue content, focusing on molecules with αααβ and ααβαααβ backbone repeat patterns. Among analogues of an 18-mer derived from the Bim BH3 domain and an 8-mer derived from secreted phospholipase-2 (sPLA2), we find that recognition by antibodies raised against the prototype α-peptide is suppressed by periodic α → β replacements. Complementary studies reveal that antibodies raised against Bim BH3- or sPLA2-derived α/β-peptides fail to recognize prototype α-peptides displaying identical side chain repertoires. Because polypeptides containing d-α-amino acid residues are of growing interest for biomedical applications, we included the enantiomer of the sPLA2-derived α-peptide in these studies; this d-peptide is fully competent as a hapten, but the resulting antibodies do not cross react with the enantiomeric peptide. Among analogues of the 9-mer CD8(+) T-cell viral epitope GP33, we observe that periodic α → β replacements suppress participation in the MHC I + peptide + T-cell receptor ternary complexes that activate cytotoxic T-lymphocytes, due in part to disruption of MHC binding.