A versatile platform for single-molecule enzymology of restriction endonuclease

Enzymes are the major players for many biological processes. Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level. Yet, these single-molecule experiments are technically difficult and generally require complicated experimental design. Here, we develop a Holliday junction (HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET (sm-FRET). We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events. Thanks to the multiple-arms structure of HJ, the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm. Therefore, it is possible to independently change the substrate arm to study different enzymes with similar functions. Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations. Moreover, this method can be easily extended to study other types of DNA-binding enzymes without too much modification of the design. We anticipate it can find broad applications in single-molecule enzymology.

Dynamic disorder in simple enzymatic reactions induces stochastic amplification of substrate

A growing amount of evidence over the last two decades points to the fact that many enzymes exhibit fluctuations in their catalytic activity, which are associated with conformational changes on a broad range of timescales. The experimental study of this phenomenon, termed dynamic disorder, has become possible thanks to advances in single-molecule enzymology measurement techniques, through which the catalytic activity of individual enzyme molecules can be tracked in time. The biological role and importance of these fluctuations in a system with a small number of enzymes, such as a living cell, have only recently started being explored. In this work, we examine a simple stochastic reaction system consisting of an inflowing substrate and an enzyme with a randomly fluctuating catalytic reaction rate that converts the substrate into an outflowing product. To describe analytically the effect of rate fluctuations on the average substrate abundance at steady state, we derive an explicit formula that connects the relative speed of enzymatic fluctuations with the mean substrate level. Under fairly general modelling assumptions, we demonstrate that the relative speed of rate fluctuations can have a dramatic effect on the mean substrate, and lead to large positive deviations from predictions based on the assumption of deterministic enzyme activity. Our results also establish an interesting connection between the amplification effect and the mixing properties of the Markov process describing the enzymatic activity fluctuations, which can be used to easily predict the fluctuation speed above which such deviations become negligible. As the techniques of single-molecule enzymology continuously evolve, it may soon be possible to study the stochastic phenomena due to enzymatic activity fluctuations within living cells. Our work can be used to formulate experimentally testable hypotheses regarding the nature and magnitude of these fluctuations, as well as their phenotypic consequences.