Fluorescent-labeled antibodies and recombinant proteins containing a fluorescent reporter are commonly used to visualize proteins in situ, but the antibodies and reporters can perturb protein structure and function. To overcome this problem, we designed minimally perturbing blue fluorescent labels (4-cyanoindol [4CN] and 4-cyano-tryptophan [4CNtrp]) that enable the direct imaging of intrinsically-fluorescent proteins. Here, we used 4CN-labeled peptides to directly image integrins on the platelet surface. 4CN has unique photophysical properties: an absorption maximum at ~325 nm, an emission maximum at ~420 nm, a large fluorescence quantum yield (&gt;0.8 in aqueous solution), a long fluorescence lifetime (ca. 13 ns), and good photostability. Previously, we described CHAMP (Computed Helical Anti-Membrane Protein) peptides that target integrin TM domains in a sequence-specific manner. Thus, the peptides anti-αIIb and anti-αv bind to the αIIb and αv TM domains, respectively, causing αIIbβ3 and αvβ3 activation. Anti-αIIb also causes αIIbβ3 clustering that we visualized using fluorescent-labeled anti-β3 monoclonal antibodies but only after the platelets were fixed to prevent antibody-induced clustering. Using solid-phase peptide synthesis, we generated CHAMP peptides labeled with 4CN at their N-termini (CN-αIIb, CN-αv, CN-β1). 4CN labeling did not prevent the ability of CN-αIIb to cause αIIbβ3-dependent platelet aggregation, CN-αv to cause αvβ3-dependent platelet adhesion to osteopontin, and CN-β1 to cause α2β1-mediated platelet adhesion to collagen. We then used 2-photon confocal imaging to detect 4CN-containing integrins on the platelet surface. Compared to a 4CN-containing random peptide that uniformly decorated the platelet surface, CN-labeled CHAMP peptides were present as a limited number of discrete foci. To confirm that these foci represent CN-peptide containing integrins, we co-stained the cells with Alexa Fluor 458-labeled integrin-specific monoclonal antibodies and found that the CN-peptide and antibody fluorescence coincided. Thus, these studies demonstrate the specific direct imaging of integrins embedded in the platelet membrane using 4CN-labeled anti-integrin TM peptides. Because 4CN can readily be incorporated into proteins and peptides with little if any structural perturbation, 4CN-labeling provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments.

Direct visualization of platelet integrins using anti-transmembrane domain peptides containing a blue fluorescent amino acid

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.