Origin of the Conformational Heterogeneity of Cardiolipin-Bound Cytochrome <i>c</i>

Interactions of cytochrome <i>c</i> (cyt <i>c</i>) with cardiolipin (CL) partially unfold the protein, activating its peroxidase function, a critical event in the execution of apoptosis. However, structural features of the altered protein species in the heterogeneous ensemble are difficult to probe with ensemble averaging. Analyses of the dye-to-heme distance distributions <i>P</i>(<i>r</i>) from time-resolved FRET (TR-FRET) have uncovered two distinct types of CL-bound cyt <i>c</i> conformations, extended and compact. We have combined TR-FRET, fluorescence correlation spectroscopy (FCS), and biolayer interferometry to develop a systematic understanding of the functional partitioning between the two conformations. The two subpopulations are in equilibrium with each other, with a submillisecond rate of conformational exchange reflecting the protein folding into a compact non-native state, as well as protein interactions with the lipid surface. Electrostatic interactions with the negatively charged lipid surface that correlate with physiologically relevant changes in CL concentrations strongly affect the kinetics of cyt <i>c</i> binding and conformational exchange. A predominantly peripheral binding mechanism, rather than deep protein insertion into the membrane, provides a rationale for the general denaturing effect of the CL surface and the large-scale protein unfolding. These findings closely relate to cyt <i>c</i> folding dynamics and suggest a general strategy for extending the time window in monitoring the kinetics of folding

Becoming a Peroxidase: Cardiolipin-Induced Unfolding of Cytochrome <i>c</i>

Interactions of cytochrome <i>c</i> (cyt <i>c</i>) with a unique mitochondrial glycerophospholipid cardiolipin (CL) are relevant for the protein’s function in oxidative phosphorylation and apoptosis. Binding to CL-containing membranes promotes cyt <i>c</i> unfolding and dramatically enhances the protein’s peroxidase activity, which is critical in early stages of apoptosis. We have employed a collection of seven dansyl variants of horse heart cyt <i>c</i> to probe the sequence of steps in this functional transformation. Kinetic measurements have unraveled four distinct processes during CL-induced cyt <i>c</i> unfolding: rapid protein binding to CL liposomes; rearrangements of protein substructures with small unfolding energies; partial insertion of the protein into the lipid bilayer; and extensive protein restructuring leading to “open” extended structures. While early rearrangements depend on a hierarchy of foldons in the native structure, the later process of large-scale unfolding is influenced by protein interactions with the membrane surface. The opening of the cyt <i>c</i> structure exposes the heme group, which enhances the protein’s peroxidase activity and also frees the C-terminal helix to aid in the translocation of the protein through CL membranes

A Photostable AIE Luminogen for Specific Mitochondrial Imaging and Tracking

Tracking the dynamics of mitochondrial morphology has attracted much research interest because of its involvement in early stage apoptosis and degenerative conditions. To follow this process, highly specific and photostable fluorescent probes are in demand. Commercially available mitochondria trackers, however, suffer from poor photostability. To overcome this limitation, we have designed and synthesized a fluorescent agent, tetraphenylethene-triphenylphosphonium (TPE-TPP), for mitochondrial imaging. Inherent from the mitochondrial-targeting ability of TPP groups and the aggregation-induced emission (AIE) characteristics of the TPE core, TPE-TPP possesses high specificity to mitochondria, superior photostability, and appreciable tolerance to environmental change, allowing imaging and tracking of the mitochondrial morphological changes in a long period of time

A Photostable AIE Luminogen for Specific Mitochondrial Imaging and Tracking

Tracking the dynamics of mitochondrial morphology has attracted much research interest because of its involvement in early stage apoptosis and degenerative conditions. To follow this process, highly specific and photostable fluorescent probes are in demand. Commercially available mitochondria trackers, however, suffer from poor photostability. To overcome this limitation, we have designed and synthesized a fluorescent agent, tetraphenylethene-triphenylphosphonium (TPE-TPP), for mitochondrial imaging. Inherent from the mitochondrial-targeting ability of TPP groups and the aggregation-induced emission (AIE) characteristics of the TPE core, TPE-TPP possesses high specificity to mitochondria, superior photostability, and appreciable tolerance to environmental change, allowing imaging and tracking of the mitochondrial morphological changes in a long period of time

A Photostable AIE Luminogen for Specific Mitochondrial Imaging and Tracking

Tracking the dynamics of mitochondrial morphology has attracted much research interest because of its involvement in early stage apoptosis and degenerative conditions. To follow this process, highly specific and photostable fluorescent probes are in demand. Commercially available mitochondria trackers, however, suffer from poor photostability. To overcome this limitation, we have designed and synthesized a fluorescent agent, tetraphenylethene-triphenylphosphonium (TPE-TPP), for mitochondrial imaging. Inherent from the mitochondrial-targeting ability of TPP groups and the aggregation-induced emission (AIE) characteristics of the TPE core, TPE-TPP possesses high specificity to mitochondria, superior photostability, and appreciable tolerance to environmental change, allowing imaging and tracking of the mitochondrial morphological changes in a long period of time

Inspecting Metal-Coordination-Induced Perturbation of Molecular Ligand Orbitals at a Submolecular Resolution

Molecular states of terpyridine ligands in supramolecular coordination assemblies were investigated by means of scanning tunneling microscopy/spectroscopy conducted at cryogenic temperature. Submolecular-resolved signals manifest that within the molecules, the empty states at the moieties that are directly involved in the coordination are downshifted, whereas the other moieties are unaffected. Theoretical calculations attribute this localized perturbation to the specific characteristics of the ligand’s orbitals; the ligand moieties possess highly localized empty states. Our results demonstrate that it is feasible to electronically modify individual moieties of ligands in supramolecular assemblies by metal coordination

Light-Enhanced Bacterial Killing and Wash-Free Imaging Based on AIE Fluorogen

The rapid acquisition of antibiotic resistance poses difficulties in the development of effective methods to eliminate pathogenic bacteria. New bactericides, especially those do not induce the emergence of resistance, are thus in great demand. In this work, we report an aggregation-induced emission fluorogen, TPE-Bac, for bacterial imaging and elimination. TPE-Bac can be readily dissolved in aqueous solution with weak emission. The presence of bacteria can turn on its emission, and thus no washing step is required in the imaging process. Meanwhile, TPE-Bac can be applied as a bactericide for elimination of bacteria. The amphiphilic TPE-Bac bearing two long alkyl chains and two positively charged amines can intercalate into the membrane of bacteria, increase membrane permeability and lead to dark toxicity. The efficiency of bacteria killing is greatly enhanced under light irradiation. TPE-Bac can serve as a photosensitizer to induce reactive oxygen species (ROS) generation, which ensures the efficient killing of bacteria. The TPE-Bac-containing agar plates can be continuously used for bacteria killing by applying light to induce ROS generation

Thiol-Reactive Molecule with Dual-Emission-Enhancement Property for Specific Prestaining of Cysteine Containing Proteins in SDS-PAGE

1-[4-(Bromomethyl)­phenyl]-1,2,2-triphenylethene (<b>2</b>) was synthesized and evaluated for specific fluorescent prestaining of proteins containing cysteine (Cys) in SDS-PAGE. The molecule showed classic aggregation-induced emission (AIE) property in protein labeling and its quantum efficiency was further enhanced upon reacting with Cys. The parameters of reaction such as labeling time and concentration of dye and reducing reagent-tris­(2-carboxyethyl)­phosphine (TCEP) were examined to obtain the optimal labeling condition. In addition to its specific labeling effect, molecule <b>2</b> also showed its advantage over traditional self-quenching dyes through labeling Cys containing BSA with different dye/Cys ratios

Superior Fluorescent Probe for Detection of Cardiolipin

Cardiolipin (CL) is a unique phospholipid found in mitochondrial inner membrane. It is a key component for mitochondrial function in both respiration and apoptosis. The level of CL is an important parameter for investigating these intracellular events and is a critical indicator of a number of diseases associated with mitochondrial respiratory functions. 10-Nonyl acridine orange (NAO) is the only fluorescent dye currently available for CL detection. However, the performance of NAO is far from satisfactory in terms of selectivity and sensitivity. In this work, we report an aggregation-induced emission-active fluorogen, TTAPE-Me, for CL detection and quantification. With improved sensitivity and excellent selectivity to CL over other major mitochondrial membrane lipids, TTAPE-Me could serve as a valuable fluorescent sensor for CL quantification. The use of TTAPE-Me for the quantification of isolated mitochondria is also demonstrated

A Ratiometric Fluorescent Probe Based on ESIPT and AIE Processes for Alkaline Phosphatase Activity Assay and Visualization in Living Cells

Alkaline phosphatase (ALP) activity is regarded as an important biomarker in medical diagnosis. A ratiometric fluorescent probe is developed based on a phosphorylated chalcone derivative for ALP activity assay and visualization in living cells. The probe is soluble in water and emits greenish-yellow in aqueous buffers. In the presence of ALP, the emission of probe changes to deep red gradually with ratiometric fluorescent response due to formation and aggregation of enzymatic product, whose fluorescence involves both excited-state intramolecular proton transfer and aggregation-induced emission processes. The linear ratiometric fluorescent response enables <i>in vitro</i> quantification of ALP activity in a range of 0–150 mU/mL with a detection limit of 0.15 mU/mL. The probe also shows excellent biocompatibility, which enables it to apply in ALP mapping in living cells