Single-enzyme kinetics with fluorogenic substrates: lessons learnt and future directions.

摘要: Single-molecule fluorescence techniques have developed into powerful tools for studying the kinetics of biological reactions at single-molecule level. Using fluorogenic substrates, enzymatic can be observed in real-time with single-turnover resolution. The turnover sequence contains all kinetic information, giving access to reaction substeps and dynamic processes such as fluctuations rate. Despite their clearly proven potential, accuracy current measurements is limited by availability substrates 1:1 stoichiometry signal-to-noise ratio measurement. In this review we summarize state-of-the-art discuss these limitations using experiments performed α-chymotrypsin an example. We are further providing overview recent efforts aimed improvement development new detection schemes. These schemes utilize nanophotonic structures zero mode waveguides or nanoantennas. Nanophotonic approaches reduce size effective volume a strategy increase ratio. believe that combination improved novel will pave way performing accurate single-enzyme biologically relevant conditions.