Photon-by-Photon Fluorescence Lifetime Analysis of Within-Burst State Transitions of Single Proteins in Lipid...

Understanding rapid, nonequilibrium dynamics of single proteins in lipid membranes is crucial but challenging. This study advances fluorescence lifetime analysis by developing a computationally efficient variational Bayesian framework for photon-by-photon hidden Markov modeling. It enables robust and accurate model selection, facilitating real-time tracking of state evolution of a molecule within a brief time frame. We applied the method to investigate nonequilibrium membrane insertion of a model peptide, revealing that unsaturated bonds in acyl chains not only merely modulate the fluidity of lipid membranes but also directly interact with transmembrane proteins, answering a long-standing question about unsaturated bonds' roles in membrane-protein interactions.