Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics

Significance

Quantification of dynamic information is fundamental to the understanding of many biological processes. However, extraction of quantitative information is often difficult due to mathematical ambiguities or the separation of conformation dynamics from photophysical processes. Here, we present a general approach that analyzes fluorescence intensity fluctuations and fluorescence lifetime information to perform a quantitative analysis of two-state systems without prior knowledge regarding the dynamics. This approach can also distinguish between photophysical on-off processes and dynamic changes between states with different fluorescence lifetime states (e.g., conformational states with different Förster resonance energy transfer (FRET) efficiencies). We demonstrate the power of this method, referred to as shrinking gate (sg)-FCS, by unravelling the mechanism of a FRET-based membrane-charge sensor.

Abstract

Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.