  Single-molecule F\"orster Resonance Energy Transfer (smFRET) is ideally
suited to resolve the dynamics of biomolecules. A significant challenge to date
is capturing and quantifying the exchange between multiple conformational
states, mainly when these dynamics occur on the sub-millisecond timescale. Many
methods for the quantitative analysis are challenged if more than two states
are involved, and the appropriate choice of the number of states in the kinetic
network is difficult. An additional complication arises if dynamically active
molecules coexist with solely static molecules in similar conformational
states. To address these problems, we developed an integrative analysis
framework that combines the information from FRET-lines, time-correlated single
photon counting, and fluorescence correlation spectroscopy. While individually,
these methodologies provide ambiguous results for quantitatively characterize
the dynamics in complex kinetic networks, the global analysis enables accurate
determination of the number of states, their kinetic connectivity, and the
transition rate constants. To challenge the potential of smFRET to study
multi-state kinetic networks, we benchmark the framework using synthetic data
of three-state systems with different kinetic connectivity and interconversion
rates.
