Protein structure by mechanical triangulation

Knowledge of protein structure is essential to understand protein function. High-resolution protein structure has so far been the domain of ensemble methods. Here, we develop a simple single-molecule technique to measure spatial position of selected residues within a folded and functional protein structure in solution. Construction and mechanical unfolding of cysteine-engineered polyproteins with controlled linkage topology allows measuring intramolecular distance with angstrom precision. We demonstrate the potential of this technique by determining the position of three residues in the structure of green fluorescent protein (GFP). Our results perfectly agree with the GFP crystal structure. Mechanical triangulation can find many applications where current bulk structural methods fail.