Co-translocational Unfolding of HP35 in MIL-101(Cr) MOF

The immobilization of enzymes in the cages of Metal-Organic Frameworks (MOF) is important in biotechnology. In this context, the mechanism of translocation of proteins through the cavities of the MOF and the roles played by confinement and MOF chemistry in giving rise to stable protein intermediates that are otherwise transiently populated in physiological environment are important questions to be addressed. Herein, these unexplored aspects are examined with Villin Headpiece (HP35) as a model protein confined within a mesopore of MIL-101(Cr) using molecular dynamics simulations. At equilibrium, the protein is located farther from the center of the cavity and closer to the MOF surface. Molecular interactions with the MOF leads to a partial unfolding of helix-1 at its N-terminus. Umbrella sampling simulations inform the range of conformations that HP35 undertakes during the translocation from one cavity to another and also provide the free energy differences at various stages of translocation. Relative to its equilibrium state within the cavity, the free energy barrier for the unfolded protein at the cage window is estimated to be 16 kcal/mol. This study of MOF-based protein conformation can also serve as a general approach to observing intermediates in folding-unfolding pathways.