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

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.