Sampling the unfolding pathways towards the signaling state of Photoactive Yellow Protein

Abstract

When receiving a signal trigger, sensor proteins undergo conformational changes resulting in the formation of a signaling state. Photoactive Yellow Protein (PYP) is a bacterial blue light sensor, 125 amino acids in size, including para-coumaric acid as a chromophore. Upon absorbing a blue-light photon, PYP undergoes a series of rearrangements to form a signaling state. The last step in this process is partial unfolding of the protein, occurring on a sub-millisecond timescale. Molecular simulation can provide detailed insight into the mechanisms underlying protein conformational changes and is complementary to experiments. Studying a protein folding reaction at atomistic resolution with conventional atomistic Molecular Dynamics (MD) is unpractical due to the long time scales involved. These long time scales originate from the presence of local free energy minima from which it is not trivial to escape. Advanced simulations enabled us to investigate the equilibrium characteristics, as well as the dynamical pathways of conformational changes linked to the formation of the signaling state of PYP. Replica exchange MD resulted in the identification of several intermediates during the light induced unfolding. Using these state as input for transition path sampling and subsequent reaction coordinate analysis led to new mechanistic insights in this conformational change. The conformational change starts with the unfolding of a helix in the chromophore binding pocket, followed by the solvent exposure of either the chromophore or glutamate at position 46. Furthermore, our simulations indicate that it is more likely that Glu46 becomes solvent exposed first. To our knowledge this is the first simulation study of unbiased dynamical pathways of a sub-millisecond timescale process of a biologically relevant protein. This work opens up the way for investigating conformational changes in other interesting systems in high detail.