Identifying the minimal sets of distance restraints for FRET-assisted protein structural modeling.

Proteins naturally occur in crowded cellular environments and interact with other proteins, nucleic acids, and organelles. Since most previous experimental protein structure determination techniques require that proteins occur in idealized, non-physiological environments, the effects of realistic cellular environments on protein structure are largely unexplored. Recently, Förster resonance energy transfer (FRET) has been shown to be an effective experimental method for investigating protein structure in vivo. Inter-residue distances measured in vivo can be incorporated as restraints in molecular dynamics (MD) simulations to model protein structural dynamics in vivo. Since most FRET studies only obtain inter-residue separations for a small number of amino acid pairs, it is important to determine the minimum number of restraints in the MD simulations that are required to achieve a given root-mean-square deviation (RMSD) from the experimental structural ensemble. Further, what is the optimal method for selecting these inter-residue restraints? Here, we implement several methods for selecting the most important FRET pairs and determine the number of pairs Nr that are needed to induce conformational changes in proteins between two experimentally determined structures. We find that enforcing only a small fraction of restraints, Nr/N≲0.08, where N is the number of amino acids, can induce the conformational changes. These results establish the efficacy of FRET-assisted MD simulations for atomic scale structural modeling of proteins in vivo. Significance: Determining protein structure in vivo is essential for understanding protein function. Most protein structures have been studied in non-physiological conditions using x-ray crystallography, NMR spectroscopy, and cryo-electron microscopy. Thus, we do not know whether the cellular environment significantly affects protein structure. We emphasize the benefits of FRET-assisted molecular dynamics simulations in characterizing protein structure in vivo at the atomic scale. We identify the minimum number of FRET pairs that can induce conformational changes in several proteins, including one that has been characterized using in-cell NMR.