Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways

Iron–sulfur (Fe–S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe–S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe–S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe–2S] and [4Fe–4S] clusters), ligand environments ([2Fe–2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe–S cluster transfer reactions are monitored between two Fdx molecules that have identical Fe–S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe–2S]–DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe–S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe–S binding proteins or other chromophores. Such advanced kinetic experiments are required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. We anticipate that this cluster detection methodology will transform the study of Fe–S cluster pathways and potentially other metal cofactor biosynthetic pathways

Quantum Dots as Förster Resonance Energy Transfer Acceptors of Lanthanides in Time-Resolved Bioassays

We report a flexible and modular design for biosensors based on exploiting semiconductor quantum dots (QDs) and their excellent Förster resonance energy transfer (FRET) acceptor properties along with the long-lived fluorescent lifetimes of lanthanide donors. We demonstrate the format’s wide application by developing a broad adenosine diphosphate (ADP) sensor with quantitative and high-throughput capabilities as a kinase/ATPase assay method. The sensor is based on a Terbium (Tb)-labeled antibody (Ab) that selectively recognizes ADP versus ATP. The Tb-labeled Ab (Ab-Tb) acts as a FRET donor to a QD, which has an ADP modified His₆-peptide conjugated to its surface via metal-affinity coordination. This strategy of using self-assembly, modified peptides to present antibody epitopes on QD surfaces is readily transferable to other assays of interest. We utilize time-resolved FRET (TR-FRET) to measure the amounts of Ab-Tb bound to the QD by looking at the emission ratio of the QD and Tb in a time-gated manner, minimizing background signal. With the addition of free ADP the antibody is competitively separated from the QD and a change in the ratiometric emission signal correlates with the free ADP concentration. The sensor obtained a detection limit below 10 nM of free ADP and quantitation limit of 35 nM ADP using 8 nM of sensor. Quantitative values were obtained for a model enzyme (glucokinase) kinetics, as well as demonstrations of the assays capability to distinguish enzyme inhibitors. We discuss future outlooks and note areas for improvement in similar design strategies

Self assembling nanoparticle enzyme clusters provide access to substrate channeling in multienzymatic cascades

Channeling between enzymes is a uniquely nanoscale phenomenon that can improve multienzymatic reaction rates. Here, the authors demonstrate that multistep enzyme cascades can self-assemble with nanoparticles into nanoclusters that access channeling and improve the underlying catalytic flux by several fold