Protonation and protein folding: Insights from single-molecule fluorescence

Protein folding and protonation are deeply interconnected. Changes in the charge states of ionizable residues alter the local electrostatics and effective pKa values, influencing how proteins navigate their conformational landscapes. These protonation events can stabilize intermediates, guide folding pathways, and introduce kinetic diversity that support functional adaptability. Thus, understanding folding-protonation coupling has become a major focus of protein science. To probe these complex dynamics, increasingly sophisticated methodologies are required. Among them, single-molecule fluorescence (SMF) techniques have emerged as particularly powerful tools, providing unprecedented resolution of folding processes coupled to protonation. Approaches such as smFRET, fluorescence lifetime analysis, and rapid pH-jump experiments make it possible to observe events in exquisite detail. They reveal intermediates that escape detection in ensemble studies, capture heterogeneous subpopulations, and uncover rare, transient events that are critical to biological function. Building on these methodological advances, several case studies illustrate how protonation shapes biological outcomes for instance, histidine-rich domains function as molecular pH sensors, viral fusion proteins exploit protonation-triggered folding to mediate host entry, and engineered bio-switches harness pKa-dependent transitions to create adaptive biomaterials. When integrated with theoretical modeling, single-molecule data provide a coherent framework that links protonation dynamics to folding mechanisms across timescales. Here, in this review, we have not only consolidated current knowledge but also identified key gaps, particularly in connecting molecular-level protonation events with cellular and pathological contexts. By bridging these dimensions, this perspective aims to inspire new strategies in biomolecular engineering, therapeutic design, and the development of responsive functional materials.