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Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to detect EVs, peptide-based curvature sensors are currently attracting an attention as a novel tool for marker-free EV detection techniques. We have previously created a curvature-sensing peptide, FAAV and applied it to develop a simple and rapid method for detection of bacterial EVs in cultured media. The method utilized the fluorescence/Förster resonance energy transfer (FRET) phenomenon to achieve the high sensitivity to changes in the EV amount. In the present study, to develop a practical and easy-to-use approach that can detect bacterial EVs by peptides alone, we designed novel curvature-sensing peptides, N-terminus-substituted FAAV (nFAAV) peptides. The nFAAV peptides exerted higher α-helix-stabilizing effects than FAAV upon binding to vesicles while maintaining a random coil structure in aqueous solution. One of the nFAAV peptides showed a superior binding affinity for bacterial EVs and detected changes in the EV amount with 5-fold higher sensitivity than FAAV even in the presence of the EV-secretory bacterial cells. We named nFAAV5, which exhibited the high ability to detect bacterial EVs, as an EV-sensing peptide. Our finding is that the coil-α-helix structural transition of the nFAAV peptides serve as a key structural factor for highly sensitive detection of bacterial EVs.

Membrane curvature formation is important for various biological processes such as cell motility, intracellular signal transmission, and cellular uptake of foreign substances. However, it remains still a challenging topic to visualize the membrane curvature formation on the cell membranes in real-time imaging. To develop and design membrane curvature-sensors, we focused on amphipathic helical peptides of proteins belonging to the Bin/Amphiphysin/Rvs (BAR) family as the starting point. BAR proteins individually have various characteristic structures that recognize different curvatures, and the derived peptides possess the potential to function as curvature sensors with a variety of recognition abilities. Peptide-based curvature sensors can have wide applications in biological research fields due to their small size, easy modification, and large production capability in comparison to protein-based sensors. In the present study, we found that an amphipathic peptide derived from sorting nexin1 (SNX1) has a curvature-recognition ability. The mutation studies of the initial peptide revealed a close correlation between the α-helicity and lipid binding ability of the peptides. In particular, the amino acids located on the hydrophobic face played a vital role in curvature recognition. The α-helix formation of the peptides was thought to serve to accommodate lipid-packing defects on the membrane surface and to maintain their binding to lipid vesicles. The structure-activity correlation found in this study have the potential to contribute to the design of peptide-based curvature sensors that will enable the capture of various life phenomena in cells.

Many membrane proteins are responsible for signaling and ionic transport necessary to maintain biological func- tions in vivo. Recently, not only conformational changes but also oligomerization have been proposed to regulate pro- tein activation. Thus, the study of membrane protein oligomerization is crucial for new drug development. The existing destructive methodologies such as immunoprecipitation, however, are not suitable to determine oligomeric states pre- cisely because of the articial aggregation of proteins after detergent solubilization. In the present study, the coiled-coil tag-probe labeling method and spectral imaging wererst combined to establish a new methodology based on ‰uores- cence resonance energy transfer (FRET) for stoichiometric analysis of the oligomeric states of membrane proteins on living cells. After validating the method for mono-, di-, and tetrameric standard membrane proteins, the oligomeric state of b2-adrenergic receptors (b2ARs) was examined to clarify its functional signicance. It was found that b2ARs could transduce cyclic adenosine 5' -monophosphate (cAMP) signals and internalize them upon treatment with ligands without showing any FRET signals. Thus, b2ARs do not form constitutive homooligomers, and homooligomerization is not necessary for the receptor function of b2ARs. Finally, the oligomeric state of full-length M2 proton-selective chan- nels of in‰uenza A virus was investigated. Although the results of X-ray crystallography and NMR studies using frag- ment peptides suggested that M2 stably forms a tetrameric channel, the full-length M2 proteins formed proton-conduct- ing dimers at neutral pH and these dimers were converted to tetramers at acidic pH, indicating that the minimal func- tional unit of the M2 channel is a dimer.

Genetic fusion of fluorescent/luminescent proteins to a target protein for specific labeling in living cells has been widely used to investigate the intracellular trafficking and oligomerization of the proteins. However, several limitations of fluorescent/luminescent proteins, such as considerable size, difficulty in controlling labeling ratio in multicolor labeling, can obscure true behaviors of the target proteins. To overcome these difficulties, post-translational labeling methods using pairs of small genetically-encodable 'tags' and synthetic 'probes' targeting the tags have been widely studied in recent years. We have developed a quick tag-probe labeling method using a high-affinity heterodimeric coiled-coil formation between the E3 tag (EIAALEK)3 attached to the target protein and the K4 probe (KIAALKE)4 labeled with a fluorophore. The labeling is cell-surface-specific and completed within 1 min, therefore suitable for monitoring oligomerization/internalization of membrane proteins on living cell surface. Taking advantage of easiness in multicolor labeling, we show that the oligomeric state of membrane proteins can be precisely analyzed based on fluorescence resonance energy transfer. By using this method, we found that β2 adrenergic receptors do not form constitutive homooligomers, and homooligomerization is not necessary for the receptor function. Furthermore, the degree of internalization of the β2 receptors following agonist stimulation was evaluated by ratiometric detection of pH decrease in endosomes.