Refolding of potato carboxypeptidase inhibitor by molecular dynamics simulations with disulfide bond constraints

The folding of the potato carboxypeptidase inhibitor (PCI) from partially unfolded conformations by the introduction of native disulfide bond constraints was studied by molecular dynamics simulations in explicit solvent. PCI consists of a globular core (Cys8 to Cys34), two flexible terminal regions (Glu1 to Ile7 and Glu35 to Gly39) and three loop regions characteristic of the family of proteins known as knottins. To generate unfolded conformations, two high temperature (600 K) simulations were performed; one with the native disulfide bonds intact (N600), and one with the disulfide bonds broken (ND600). For comparison purposes, two simulations at 300 K were done; one with the native disulfide bonds (N300), and one with the disulfide bonds broken (ND300). The N300 simulation reached an energetic equilibrium within a few picoseconds and maintained a stable structure during the 500 ps simulation. The three other simulations led to partial unfolding. The largest changes were observed in ND600 simulation with an rms deviation of over 5 A and radius of gyration 12.5% larger than the crystal structure value. Six structures from the ND600 simulation and one from the N600 simulation were used as starting structures for nine refolding simulations with somewhat different protocols for reforming the native disulfide bonds; in all cases the disulfides were reformed at 600 K and the temperature was decreased to 300 K for equilibration of the folded structures. Except for one structure that was significantly misfolded (final rms of 6.64 A with respect to N300), the other folding simulations recovered the native simulation structure (N300) to within rms differences ranging from 1.8 to 3.2 A for the main-chain of the core, relative to the N300, the X-ray and the NMR structures. Of particular interest is the internal and overall refolding behavior of the three loop regions. The more unfolded starting structures led to smaller rms values for the folded structures. Several energetic and solvation models were used to evaluate the X-ray, NMR, N300 and refolded structures. Although most models can distinguish the X-ray, NMR and N300 from the refolded structures, there is no correlation between the rms values of the latter and their estimated stability. Implications of the present results for protein folding by simulations and database search methods are discussed.