Multiply Perturbed Response: A Computational Protocol to Identify Cooperative Allosteric Residue Combinations Driving Protein Conformational Transitions.

Protein function often depends on dynamic conformational transitions driven by external factors or molecular interactions. Understanding the allosteric mechanisms underlying these transitions is essential for mechanistic insight into protein function. Molecular dynamics (MD) simulations are widely used to study protein dynamics; however, capturing large-scale, rare transitions is computationally expensive. To address this, we previously developed Perturbation Response Scanning (PRS), based on elastic network models and linear response theory, but PRS is limited in capturing collective effects because it perturbs one residue at a time. Here, we present Multiply Perturbed Response (MPR), which extends PRS by applying simultaneous perturbations to multiple residues to identify allosteric residue combinations that drive conformational transitions. This protocol provides a workflow for structure preparation, displacement, and covariance-matrix calculations, overlap analysis, and visualization. It can be applied to static structures or trajectories from MD simulations, requiring initial and final protein structures as the main inputs and an optional MD trajectory for trajectory-based analysis. The main outputs are residue combinations that maximize overlap,