Org Biomol Chem

A new fluorescent label N-[4'-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-beta-alanine () 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 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

Sensing peptide–oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein

We present a new methodology for site-specific sensing of peptide–oligonucleotide (ODN) interactions using a solvatochromic fluorescent label based on 3-hydroxychromone (3HC). This label was covalently attached to the N-terminus of a peptide corresponding to the zinc finger domain of the HIV-1 nucleocapsid protein (NC). On interaction with target ODNs, the labeled peptide shows strong changes in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from the bulk water by the ODN bases. Remarkably, this two-color response depends on the ODN sequence and correlates with the 3D structure of the corresponding complexes, suggesting that the 3HC label monitors the peptide–ODN interactions site-specifically. By measuring the two-color ratio, we were also able to determine the peptide–ODN-binding parameters and distinguish multiple binding sites in ODNs, which is rather difficult using other fluorescence methods. Moreover, this method was found to be more sensitive than the commonly used steady-state fluorescence anisotropy, especially in the case of small ODNs. The described methodology could become a new universal tool for investigating peptide–ODN interactions

A Four-Amino Acid Linker between Repeats in the α‑Synuclein Sequence Is Important for Fibril Formation

α-Synuclein is a 140-amino acid protein that can switch conformation among intrinsically disordered in solution, helical on a membrane, and β-sheet in amyloid fibrils. Using the fluorescence of single-tryptophan mutants, we determined the immersion of different regions of the protein into lipid membranes. Our results suggest the presence of a flexible break close to residues 52–55 between two helical domains. The four-amino acid linker is not necessary for membrane binding but is important for fibril formation. A deletion mutant lacking this linker aggregates extremely slowly and slightly inhibits wild-type aggregation, possibly by blocking the growing ends of fibrils

Non-uniform self-assembly:On the anisotropic architecture of α-synuclein supra-fibrillar aggregates

Although the function of biopolymer hydrogels in nature depends on structural anisotropy at mesoscopic length scales, the self-assembly of such anisotropic structures in vitro is challenging. Here we show that fibrils of the protein α-synuclein spontaneously self-assemble into structurally anisotropic hydrogel particles. While the fibrils in the interior of these supra-fibrillar aggregates (SFAs) are randomly oriented, the fibrils in the periphery prefer to cross neighboring fibrils at high angles. This difference in organization coincides with a significant difference in polarity of the environment in the central and peripheral parts of the SFA. We rationalize the structural anisotropy of SFAs in the light of the observation that αS fibrils bind a substantial amount of counterions. We propose that, with the progress of protein polymerization into fibrils, this binding of counterions changes the ionic environment which triggers a change in fibril organization resulting in anisotropy in the architecture of hydrogel particles

Association of α-Synuclein with Lipid Vesicles : Stopped-Flow Kinetics of Concerted Binding and Conformational Change

Alpha-synuclein (AS), a 140aa intrinsically disordered protein, self-associates into oligomeric forms and aggregates into amyloid fibrils in Parkinson's disease. Certain mutations affect these processes and accelerate disease pathogenesis. The physiological roles of AS are a matter of speculation. Membrane binding is undoubtedly involved and the protein acquires α-helical structure in the process (1).We have studied the thermodynamics and kinetics of AS-membrane association utilizing vesicles (SUVs) of differing composition. Functionally neutral single cysteine mutants of AS were labeled with a polarity sensitive excited-state intramolecular proton transfer (ESIPT) probe (MFE). Double cysteine mutants were labeled with a FRET pair (Alexa Fluor488, Alexa Fluor568) at a series of selected positions in the primary sequence. Kinetic studies were conducted by stopped-flow, using 5-20 nM protein concentrations and increasing levels of SUVs (generally 20-200 µM) Signal changes indicative of membrane association were observed: increased intensity and shape change of dual band ESIPT emission, and altered FRET with the Alexa dyes. The analysis revealed a two-step reaction sequence in the time range <10 s. We attribute the first step to binding, and from the dependence on lipid concentration determined the second order rate constants and corresponding spectroscopic parameters. The second concentration independent step (1-10 s range) presumably arises from conformational changes in the protein (α-helix formation) and its accommodation to or perturbation of the lipid microenvironment (ESIPT dye).Accompanying thermodynamic measurements led to estimates of dissociation constants as a function of membrane composition, charge, and shape (SUVs, LUVs). A new experimental protocol (slopes), implemented in a microplate reader, circumvented technical problems usually manifested in titrations of protein with lipid.Instituto de Investigaciones Bioquímicas de La Plat

Highly solvatochromic 7-aryl-3-hydroxychromones

Introduction of the dialkylaminophenyl group in position 7 of 3-hydroxychromone changes the orientation of the excited-state dipole moment and leads to superior solvatochromic properties (>170 nm emission shift in aprotic media). The excited-state intramolecular proton-transfer (ESIPT) reaction of 7-aryl-3-hydroxychromones is almost completely inhibited in most solvents. Methylation of the 3-OH abolishes ESIPT completely and also leads to improved photostability. The probes exhibit a ∼100-fold increase in fluorescence intensity and large Stokes shifts upon binding to membranes, reflecting differences in membrane phase and charge by a >40 nm spread in the emission band position.Fil: Giordano, Luciana. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Shvadchak, Volodymyr V.. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Fauerbach, Jonathan Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Jares, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Jovin, Thomas M.. Max Planck Institute For Biophysical Chemistry; Alemani

Fibril Breaking Accelerates α‑Synuclein Fibrillization

The formation of amyloid fibrils of α-synuclein (αSyn), the key protein in Parkinson’s disease, is an autocatalytic process that is seeded by mature αSyn fibrils. Based on systematic measurements of the dependence of the fibril growth rate on the concentrations of monomers and preformed fibrillar seeds, we propose a mechanism of αSyn aggregation that includes monomer binding to fibril ends and secondary nucleation by fibril breaking. The model explains the increase of the αSyn aggregation rate under shaking conditions and the exponential increase in the fraction of fibrillar protein at the initial stages of αSyn aggregation. The proposed autocatalytic mechanism also accounts for the high variability in the aggregation lag time. The rate constant of monomer binding to the ends of fibrils, <i>k</i>_<i>+</i> ≈ 1.3 mM^–1 s^–1, was estimated from the aggregation rate and previously reported average fibril lengths. From the aggregation rates at low concentrations the binding of monomeric αSyn to fibrils was found to be almost irreversible, with an equilibrium dissociation constant (<i>K</i>_d) smaller than 3 μM