Dual-fluorescence L-amino acid reports insertion and orientation of melittin peptide in cell membranes.

Monitoring insertion and orientation of peptides in situ on cell membranes remains a challenge. To this end, we synthesized an l-amino acid (AFaa) containing a dual-fluorescence dye of the 3-hydroxyflavone family, as a side chain. In contrast to other labeling approaches using a flexible linker, the AFaa fluorophore, introduced by solid phase synthesis into desired position of a peptide, is attached closely to its backbone with well-defined orientation, and, therefore, could reflect its localization in the membrane. This concept was validated by replacing the leucine-9 (L9) and tryptophan-19 (W19) residues by AFaa in melittin, a well-studied membrane-active peptide. Due to high sensitivity of AFaa dual emission to the environment polarity, we detected a much deeper insertion of L9 peptide position into the bilayer, compared to the W19 position. Moreover, using fluorescence microscopy with a polarized light excitation, we found different orientation of AFaa at L9 and W19 positions of melittin in the bilayers of giant vesicles and cellular membranes. These results suggested that in the natural membranes, similarly to the model lipid bilayers, melittin is preferentially oriented parallel to the membrane surface. The developed amino acid and the proposed methodology will be of interest to study other membrane peptides.

Two-color fluorescent l-amino acid mimic of tryptophan for probing peptide-nucleic acid complexes.

Non-natural amino acids are important tools for site-selective probing of peptide properties and interactions. Here, for the first time a fluorescent l-amino acid, exhibiting excited-state intramolecular proton transfer (ESIPT) and hydration-sensitive dual emission, was synthesized. It is an analogue of l-tryptophan bearing a slightly larger 2-(2-furyl)-3-hydroxychromone aromatic moiety instead of indole. This new amino acid was incorporated through solid-phase synthesis into NC(11-55), the zinc finger domain of the HIV-1 nucleocapsid protein, that exhibits potent nucleic acid chaperone properties. It was substituted for the Trp37 and Ala30 residues, located in the distal finger motif and the linker between the fingers of NC(11-55), respectively. Though the highly conserved Trp37 residue plays a key role in NC(11-55) structure and activity, its substitution for the new fluorescent analogue preserved the folding, the nucleic acid binding and chaperone activity of the peptide, indicating that the new amino acid can conservatively substitute Trp residues. In the presence of oligonucleotides, the Trp37-substituted peptide, but not the Ala30 variant, showed strong changes of the dual emission corresponding to local dehydration. The results are in line with NMR data, suggesting that the fluorescent amino acid interacts similarly to Trp37 with the nucleobases and is thus screened from water. Due to the exceptional sensitivity of its ESIPT fluorophore to hydration in highly polar environment, the new amino acid appears as a promising tool for substituting Trp residues and site-selectively investigating peptide-nucleic acid complexes.