Atomic Level Characterization Of Millisecond-Time Scale Protein Motions Through A Combined Molecular Simulations And Nmr Approach

Advances in biomolecular sciences are closely linked to our ability to chart the energy landscapes of biomolecules with atomic details. Here we validate a new paradigm to characterise thermodynamics and kinetics of millisecond timescale conformational transitions between ground state and transient excited states in the enzyme cyclophilin A (CypA). We describe a novel methodology that combines molecular dynamics simulations and Markov State modelling with NMR measurements to provide atomic-level insights into the nature of CypA transient conformational states. The computed conformational ensembles also enabled the predictive design and experimental validation of a single-site mutant that dramatically perturbs millisecond timescale loop motions, converting a CypA excited state into the ground state. The resulting models open up new horizons for targeting CypA with inhibitors and pave the way towards rational design of protein energy landscapes for protein engineering and drug discovery purposes.