Prediction of the translocation kinetics of a protein from its mechanical properties

Abstract

Proteins are actively unfolded to pass through narrow channels in macromolecular complexes that catalyze protein translocation and degradation. Catalyzed unfolding shares many features that characterize the mechanical unfolding of proteins using the atomic force microscope (AFM). However, simulations of unfolding induced by the AFM and when a protein is translocated through a pore suggest that each process occurs by distinct pathways. The link, if any, between each type of unfolding, therefore, is not known. We show that the mechanical unfolding energy landscape of a protein, obtained using an atomistic molecular model, can be used to predict both the relative mechanical strength of proteins when unfolded using the AFM and when unfolded by translocation into a pore. We thus link the two processes and show that the import rate through a pore not only depends on the location of the initiation tag but also on the mechanical properties of the protein when averaged over all the possible geometries that are relevant for a given translocation initiation site.

FIGURE 1

(a) AFM single molecule experiments can pull pairs of atoms apart under a constant loading force F. By repeating the same experiment a number of times, one may determine the unfolding rate for a given residue pair and force. (b) A simple steric model of a pore. Assuming that the protein unravels as a consequence of being dragged through the pore, a special role will be played by the residues that contact the rim of the pore. Their identity depends not only on the site of the translocation initiation site but also on a possible partial unraveling of the protein in the region of the initiation site.

FIGURE 2

Mechanical unfolding landscape of I27 generated by estimating unfolding rates pulling apart all pairs of residues at a constant force of 150 pN, T = 300 K. Yellow to blue colors denote high to weak mechanical resistance; black denotes pairs not pulled. Scale is in ps⁻¹.

FIGURE 3

Unfolding rates measured when pulling I27 by a single residue through a pore (black line). The translocation rate through the pore is related to the mechanical landscape when averaged over suitable geometries. The blue line is the unfolding time averaged over all possible residue pairs; the red from averaging over all pairs with c_ij determined as described in the text.