Single Photon smFRET. II. Application to Continuous Illumination

Here we adapt the Bayesian nonparametrics (BNP) framework presented in the first companion manuscript to analyze kinetics from single photon, single molecule Förster Resonance Energy Transfer (smFRET) traces generated under continuous illumination. Using our sampler, BNP-FRET, we learn the escape rates and the number of system states given a photon trace. We benchmark our method by analyzing a range of synthetic and experimental data. Particularly, we apply our method to simultaneously learn the number of system states and the corresponding kinetics for intrinsically disordered proteins (IDPs) using two-color FRET under varying chemical conditions. Moreover, using synthetic data, we show that our method can deduce the number of system states even when kinetics occur at timescales of interphoton intervals. Why It Matters In the first companion manuscript of this series, we developed new methods to analyze noisy smFRET data. These methods eliminate the requirement of a priori specifying the dimensionality of the physical model describing a molecular complex’s kinetics. Here, we apply these methods to experimentally obtained datasets with samples illuminated by time-invariant laser intensities. In particular, we study interactions of IDPs. ### Competing Interest Statement The authors have declared no competing interest.