Label-Free Single Protein Dynamics Revealed by Metasurface-Enhanced Raman Spectroscopy

A major challenge in biomedical science is a direct observation of conformational dynamics in single proteins, interacting with drug molecules, ligands, and other biomolecules. This limitation prevents access to free-energy landscapes that are fundamental to drug binding and transport and regulation of protein functions. Existing single-molecule experimental techniques rely on labels or tethering that can perturb the free-energy landscape or lack structural resolution. Here, we present an engineered plasmonic metasurface platform that enables label-free mapping of the conformational free-energy landscapes of individual proteins using metasurface-enhanced Raman spectroscopy. With single-molecule sensitivity, we resolve the predominant secondary structures adopted by bovine serum albumin protein while interacting with various drug-like functional groups in different pH conditions. We construct the free-energy landscapes and the transition pathways that reveal interconversion probabilities between α helix and either β sheet or random coil. These results reveal insights into the cooperative role of electrostatic interactions and chemical functionality on the conformational energy landscape. Our approach establishes a platform to study protein function, drug screening and testing, and protein-ligand interactions.