What is it about?
Proteins are tiny molecular machines that must fold into the correct shape to work properly. This folding process is not a simple jump from an unfolded chain to a final structure. Proteins often pass through short-lived intermediate shapes, but these are difficult to see because they appear only briefly. In this study, we developed NEXT-FRET, a single-molecule method that follows proteins as they fold in solution, under near-native conditions. Using maltose-binding protein as a model, we detected a previously hidden folding intermediate and showed that a signal peptide and cellular chaperones can redirect the folding route by stabilizing different intermediate states. In simple terms, NEXT-FRET reconstructs a molecular “movie” of protein folding and reveals steps that conventional approaches can miss.
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Why is it important?
Most methods describe proteins mainly by their final structures, but biological function often depends on the routes proteins take, the detours they explore, and the short-lived states they visit along the way. These transient states can guide correct folding, slow folding down, or trap proteins in non-productive forms linked to malfunction and disease. NEXT-FRET makes it possible to observe these hidden states in freely diffusing proteins, without attaching them to surfaces, separating them in microfluidic devices, or using conditions that may disturb their natural behavior. This opens a way to study challenging systems such as aggregation-prone protein precursors and proteins interacting with chaperones. More broadly, the approach can help us understand protein folding, misfolding, cellular quality control, and future strategies to target dynamic protein states in biotechnology and medicine.
Perspectives
For me, this study is exciting because it shows how much biology remains hidden when we look only at final protein structures. Proteins are dynamic, and their behavior is shaped by fleeting states that are often invisible to classical experiments. NEXT-FRET gives us a practical way to observe these states in solution and to ask how cells guide proteins toward productive folding or trap them in alternative routes. I also see this work as a strong example of interdisciplinary science: protein biophysics, advanced optics, and mathematical/statistical modelling were all essential. The collaboration between IMBB-FORTH, IESL-FORTH and IACM-FORTH, initiated by the FORTH Synergy Grant, was critical for turning a difficult biological question into a new experimental and analytical framework.
Giorgos Gouridis
Institute of Molecular Biology and Biotechnology (IMBB-FORTH)-Hellas
Read the Original
This page is a summary of: NEXT-FRET maps nonequilibrium rerouting of
Escherichia coli
maltose-binding protein folding by its signal peptide and chaperones, Proceedings of the National Academy of Sciences, May 2026, Proceedings of the National Academy of Sciences,
DOI: 10.1073/pnas.2529979123.
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