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The fluorescent reporters commonly used to visualize proteins can perturb both protein structure and function. Recently, we found that 4-cyanotryptophan (4CN-Trp), a blue fluorescent amino acid, is suitable for one-photon imaging applications. Here, we demonstrate its utility in two-photon fluorescence microscopy by using it to image integrins on cell surfaces. Specifically, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4-cyanoindole (4CNI) at their N-termini to image integrins on cell surfaces. CHAMP (computed helical anti-membrane protein) peptides spontaneously insert into membrane bilayers to target integrin transmembrane domains and cause integrin activation. We found that 4CNI labeling did not perturb the ability of CHAMP peptides to insert into membranes, bind to integrins, or cause integrin activation. We then used two-photon fluorescence microscopy to image 4CNI-containing integrins on the surface of platelets. Compared to a 4CNI-labeled scrambled peptide that uniformly decorated cell surfaces, 4CNI-labeled CHAMP peptides were present in discrete blue foci. To confirm that these foci represented CN peptide-containing integrins, we co-stained platelets with integrin-specific fluorescent monoclonal antibodies and found that CN peptide and antibody fluorescence coincided. Because 4CNI can readily be biosynthetically incorporated into proteins with little if any effect on protein structure and function, it provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. In addition, these results clearly demonstrate that the two-photon excitation cross section of 4CN-Trp is sufficiently large to make it a useful two-photon fluorescence reporter for biological applications.

Computationally designed transmembrane α-helical peptides (CHAMP) have been used to compete for helix-helix interactions within the membrane, enabling the ability to probe the activation of the integrins αIIbβ3 and αvβ3. Here, this method is extended towards the design of CHAMP peptides that inhibit the association of the α5β1 transmembrane (TM) domains, targeting the Ala-X3-Gly motif within α5. Our previous design algorithm was performed alongside a new workflow implemented within the widely used Rosetta molecular modeling suite. Peptides from each computational approach activated integrin α5β1 but not αVβ3 in human endothelial cells. Two CHAMP peptides were shown to directly associate with an α5 TM domain peptide in detergent micelles to a similar degree as a β1 TM peptide does. By solution-state nuclear magnetic resonance, one of these CHAMP peptides was shown to bind primarily the integrin β1 TM domain, which itself has a Gly-X3-Gly motif. The second peptide associated modestly with both α5 and β1 constructs, with slight preference for α5. Although the design goal was not fully realized, this work characterizes novel CHAMP peptides activating α5β1 that can serve as useful reagents for probing integrin biology.

Lateral transmembrane (TM) helix-helix interactions between single-span membrane proteins play an important role in the assembly and signaling of many cell-surface receptors. Often, these helices contain two highly conserved yet distinct interaction motifs, arranged such that the motifs cannot be engaged simultaneously. However, there is sparse experimental evidence that dual-engagement mechanisms play a role in biological signaling. Here, we investigate the function of the two conserved interaction motifs in the TM domain of the integrin β3-subunit. The first motif uses reciprocating "large-large-small" amino acid packing to mediate the interaction of the β3 and αIIb TM domains and maintain the inactive resting conformation of the platelet integrin αIIbβ3. The second motif, S-x3-A-x3-I, is a variant of the classical "G-x3-G" motif. Using site-directed mutagenesis, optical trap-based force spectroscopy, and molecular modeling, we show that S-x3-A-x3-I does not engage αIIb but rather mediates the interaction of the β3 TM domain with the TM domain of the αv-subunit of the integrin αvβ3. Like αIIbβ3, αvβ3 on circulating platelets is inactive, and in the absence of platelet stimulation is unable to interact with components of the subendothelial matrix. However, disrupting any residue in the β3 S-x3-A-x3-I motif by site-directed mutations is sufficient to induce αvβ3 binding to the αvβ3 ligand osteopontin and to the monoclonal antibody WOW-1. Thus, the β3-integrin TM domain is able to engage in two mutually exclusive interactions that produce alternate α-subunit pairing, creating two integrins with distinct biological functions. [ABSTRACT FROM AUTHOR]