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Abrupt reduction/resumption of thermal fluctuations of a force probe has been used to identify association/dissociation events of protein-ligand bonds. We show that off-rate of molecular dissociation can be estimated by the analysis of the bond lifetime, while the on-rate of molecular association can be estimated by the analysis of the waiting time between two neighboring bond events. However, the analysis relies heavily on subjective judgments and is time-consuming. To automate the process of mapping out bond events from thermal fluctuation data, we develop a hidden Markov model (HMM)-based method.

An HMM-based algorithm for evaluating rates of receptor–ligand binding kinetics from thermal fluctuation data