Fluorescent amino acid undergoing excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions.

Fluorescent amino acids bearing environment-sensitive fluorophores are highly valuable tools for site-selective probing of peptide/ligand interactions. Herein, we synthesized a fluorescent l-amino acid bearing the 4'-methoxy-3-hydroxyflavone fluorophore (M3HFaa) that shows dual emission, as a result of an excited state intramolecular proton transfer (ESIPT). The dual emission of M3HFaa was found to be substantially more sensitive to hydration as compared to previous analogues. By replacing the Ala30 and Trp37 residues of a HIV-1 nucleocapsid peptide, M3HFaa was observed to preserve the peptide structure and functions. Interaction of the labeled peptides with nucleic acids and lipid vesicles produced a strong switch in their dual emission, favoring the emission of the ESIPT product. This switch was associated with the appearance of long-lived fluorescence lifetimes for the ESIPT product, as a consequence of the rigid environment in the complexes that restricted the relative motions of the M3HFaa aromatic moieties. The strongest restriction and thus the longest fluorescence lifetimes were observed at position 37 in complexes with nucleic acids, where the probe likely stacks with the nucleobases. Based on the dependence of the lifetime values on the nature of the ligand and the labeled position, two-photon fluorescence lifetime imaging was used to identify the binding partners of the labeled peptides microinjected into living cells. Thus, M3HFaa appears as a sensitive tool for monitoring site selectively peptide interactions in solution and living cells.

Monitoring penetratin interactions with lipid membranes and cell internalization using a new hydration-sensitive fluorescent probe.

A new fluorescent label N-[4'-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-ß-alanine (7AF) was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The 7AF label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms.

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.

Quantification of local hydration at the surface of biomolecules using dual-fluorescence labels.

By using four labels of the 3-hydroxyflavone family displaying selective sensitivity to hydrogen bond (HB) donors and poor response to other polar molecules, we developed an approach for measuring local water concentration [H(2)O](L) (or partial volume of water: W(A) = [H(2)O](L)/55.6) in the label surrounding both in solvent mixtures and in biomolecules by the intensity ratio of two emissive forms of the label, N*/T*. Using a series of binary water/solvent mixtures with limited preferential solvation effects, a linear dependence of log(N*/T*) on the local concentration of HB donor was obtained and then used as a calibration curve for estimating the W(A) values in the surroundings of the probes conjugated to biomolecules. By this approach, we estimated the hydration of the labels in different peptides and their complexes with DNAs. We found that W(A) values for the label at the peptide N-terminus are lower (0.63-0.91) than for free labels and depend strongly on the nature of the N-terminal amino acid. When complexed with different DNAs, the estimated hydration of the labels conjugated to the labeled peptides was much lower (W(A) = 0-0.47) and depended on the DNA nature and linker-label structure. Thus, the elaborated method allows a site-specific evaluation of hydration at the surface of a biomolecule through the determination of the partial volume of water. We believe the developed procedure can be successfully applied for monitoring hydration at the surface of any biomolecule or nanostructure.

Improved hydration-sensitive dual-fluorescence labels for monitoring peptide-nucleic acid interactions.

Environmentally sensitive labels constitute a new, attractive tool for monitoring biomolecular interactions. 3-Hydroxychromone derivatives are of particular interest because they undergo excited-state intramolecular proton transfer (ESIPT) showing dual emission highly sensitive to environmental hydration. To overcome the drawbacks of the previously developed label for sensing protein-DNA interactions based on 2-furanyl-3-hydroxychromone (FC), a series of hydration-sensitive labels based on 3-hydroxy-4'-methoxyflavone have been synthesized. As compared to FC, the new labels display higher sensitivity of the ratio of their two emission bands (N*/T*) to solvent polarity and H-bond donor ability, as well as higher fluorescence quantum yields in water. Moreover, they show higher pK(a) values of their 3-hydroxyl group, allowing their application at neutral pH without interference of anionic forms. To illustrate the applications of these labels, we covalently coupled them to the N-terminus of the Tat(44-61) peptide that corresponds to the basic domain of the HIV-1 Tat protein. This coupling did not modify the nucleic acid chaperone properties of the peptide. Binding of oligonucleotides of varying length, sequence, and strandedness to the labeled peptides induced dramatic change in the N*/T* ratio of their two emission bands. This change indicated that the level of probe hydration in the peptide/oligonucleotide complexes decreases in the following order: short ssDNAs ≫ long ssDNAs > DNA hairpins > dsDNAs. The level of probe hydration was related to the ability of the probe to stack with the DNA bases or base pairs in the various complexes. The changes in the N*/T* ratio upon interaction of the labeled Tat peptides with DNA were about 3-fold larger with the new probes as compared to the parent FC label, in line with the higher sensitivity of the new probes to the environment. One of these labels, presenting the most compact geometry, showed the highest sensitivity, probably due to its optimal stacking with the DNA bases. Thus, the new hydration-sensitive labels appear as improved highly sensitive tools to site-selectively monitor the binding of peptides to oligonucleotides and nucleic acids.