On the unfolding of α‐lytic protease and the role of the pro region

Abstract

Molecular dynamics simulations of alpha-lytic protease (alphaLP) alone and complexed with its pro region (PRO) are performed to understand the origin of its high unfolding (and folding) barrier when it is alone and how the pro region lowers this barrier. At room temperature, alphaLP exhibits lower dynamic fluctuations than alpha-chymotrypsin. Simulation of PRO alone led to reorientation of its N terminal helix and collapse to a more compact state. A model for the uncleaved proenzyme was built and found to be stable in the time scale of the simulations. Energetic analysis suggests that the origin of strain in the uncleaved proenzyme compared with the cleaved complex is in the intramolecular backbone electrostatic interactions of the cleaved strand. In high temperature simulations, the interaction of the long beta hairpin of the enzyme with the C terminal beta sheet of PRO is among the most stable in the complex and a likely "nucleation site" for folding. In the course of unfolding, the C terminal tail of PRO is sometimes observed to intervene between the long hairpin and the aspartate loop of the enzyme, perhaps thereby lowering the energy barrier for separation of the two hairpins. Tighter interactions at the interface between the enzyme and its pro region are also occasionally observed, providing an additional mechanism for unfolding catalysis. Simulations of a mutant enzyme where the buried ion pair residues R102 and D142 were replaced by W and L, respectively, did not display any distinguishable behavior compared with the wild type.

Conformational polymorphism of wild‐type and mutant prion proteins: Energy landscape analysis

“…This model uses neutralized ionic side‐chains and a linear distance‐dependent dielectric function to simulate the shielding effects of water on electrostatic interaction, eliminating long‐range electrostatic interactions. The EEF1 model has been shown to give good results for the folding42–44 and unfolding45, 46 processes of various proteins and is able to discriminate between native and misfolded protein structures 47–49. Moreover, simulations using this model have been shown to yield a good agreement with explicit solvent simulations.…”

Contributions to the binding free energy of ligands to avidin and streptavidin

“…It yields modest deviations from crystal structures upon MD simulations at room temperature, unfolding pathways in agreement with explicit solvent simulations, and it discriminates native conformations from misfolded decoys 50. It has been used to determine the folding free energy landscape of a β‐hairpin,51 exploration of partially unfolded states of α‐lactalbumin,52 studies of protein unfolding,53–56 identification of stable building blocks in proteins,57 and analysis of the energy landscape of polyalanine 58…”

Thermal unfolding molecular dynamics simulation of Escherichia coli dihydrofolate reductase: Thermal stability of protein domains and unfolding pathway

“…All ionizable side‐chains are neutralized in this model, and the solvation term is parameterized to reproduce the solvation energy of small model compounds. The ability of this model to attain a global balance between the intramolecular protein–protein interactions and the protein–solvent interactions in protein simulation was shown by its ability to reproduce root‐mean‐square (RMSDs) deviations similar to those in explicit solvent simulation for stable native protein conformations21 as well as its recent application in protein unfolding studies, which yielded results comparable to those of explicit solvent simulations 10, 24…”

“…21 for a detailed discussion). Finally, although the rate at which unfolding takes place is expected to be shorter than that which would be observed in explicit solvent model simulations due to the lack of friction and third body collision effects with the actual solvent atoms, the unfolding events are expected to be similar, provided that sufficient sampling of the unfolding pathway is taken into account 10, 24…”