A new study accepted for publication in the Proceedings of the National Academy of Sciences of the USA introduces NEXT-FRET, a single-molecule approach that allows scientists to follow how proteins fold in solution and to detect short-lived molecular states that are normally missed.
Proteins are the tiny molecular machines that perform most of the work inside living cells. To function, each protein must fold into the correct three-dimensional shape. This process is not a simple jump from an unfolded chain to a final structure. Instead, proteins often pass through fleeting intermediate shapes. These transient states are important because they can guide correct folding, slow it down, or trap the protein in non-productive routes linked to malfunction and disease.
The study focuses on maltose-binding protein, a well-established model for understanding protein folding. Using NEXT-FRET, the researchers detected a previously hidden folding intermediate in freely diffusing protein molecules, under near-native conditions. They further showed that a signal peptide and molecular chaperones, both cellular factors that assist newly made proteins, can redirect the folding route by stabilizing different intermediate states.
This work is also an example of strong cross-disciplinary research within FORTH. The Dynamic Structural Biology Laboratory at IMBB-FORTH contributed expertise in protein biophysics and single-molecule folding. Researchers at IESL-FORTH contributed advanced biophotonics, optical instrumentation and microscope infrastructure. The IACM-FORTH team developed the mathematical and statistical framework needed to extract time-dependent information from complex single-molecule measurements. This collaboration was supported by the Theodore Papazoglou FORTH Synergy Grant, highlighting how institutional synergy can create methods that no single discipline could develop alone.
In simple terms, NEXT-FRET reconstructs a molecular “movie” of folding. Unlike many classical approaches, it does not require proteins to be attached to surfaces or separated in microfluidic devices, allowing folding to be followed in solution. This makes the method particularly useful for challenging biological systems, including aggregation-prone protein precursors and proteins interacting with chaperones.
Beyond this specific protein, the study provides a broader conceptual advance: it shows that biological molecules should not be understood only by their final structures, but also by the routes, detours and short-lived states they explore on the way. Such information is essential for understanding protein folding, misfolding, cellular quality control and future strategies to target dynamic protein states in biotechnology and medicine.
The study was carried out by Chara Sarafoglou and Andreas Kofidis, who contributed equally, together with Marijn de Boer, Mikis Mylonakis, Kostas Mavrakis and Giannis Zacharakis. The corresponding authors are Yannis Pantazis and Giorgos Gouridis. The work involved researchers from FORTH, the University of Crete, the University of Groningen and Kymatonics.
