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

Abstract 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.