NEXT-FRET: A solution-based smFRET platform to resolve folding intermediates under native conditions

Folding intermediates are promising therapeutic targets in protein misfolding, bacterial virulence, and drug discovery. Yet, directly observing these transient, non-equilibrium states under physiological conditions remains challenging. Here, we introduce NEXT-FRET (Non-Equilibrium miXTure modeling of smFRET), a solution-based single-molecule FRET (smFRET) platform integrating accessible instrumentation and a time-variant Gaussian Mixture Model (tvGMM) to identify folding intermediates without microfluidics, surface tethering, or denaturants. Using maltose-binding protein (MBP) and its biologically relevant precursor (pre-MBP) for benchmarking and validation, we uncover previously elusive intermediates, including a long-hypothesized closed conformation undetected by existing single-molecule and ensemble methods. Furthermore, we demonstrate that chaperones modulate folding landscapes by stabilizing distinct intermediates, some effectively arresting folding progression; a mechanism increasingly recognized as a therapeutic strategy for targeted protein inactivation. Broadly applicable beyond folding, NEXT-FRET provides a generalizable, high-resolution analytical method for resolving transient biomolecular intermediates, offering a screening-compatible platform in biotechnology and elucidating proteostasis collapse in conformational diseases.