(276c) Folding, Misfolding and Aggregation of β-Protein: Langevin Dynamics Simulations

In the manufacture of recombinant proteins, aggregates formed during protein folding process are deleterious for active recovery of target protein. While the protein aggregation in solution is observed in many experiments, the underlying mechanism remains poorly understood, especially at molecular level. In this work, we investigate the formation of amorphous protein aggregates by Langevin dynamics simulation of systems containing 10 ~ 100 model β-barrel proteins, using Gromacs as platform. It was found that at all temperatures, in response to the increase in protein concentration, the folding yield increases firstly and then decreases, i.e., a suitable concentration exists for optimal folding. In addition, the suitable temperature for protein folding shifts to a lower temperature. Misfolded, extended and native conformations were identified in the aggregates and the product spectrum was as a function of temperature and protein concentration. Misfolded form of protein becomes predominant at a low temperature and a dilute protein concentration. At a high protein concentration, however, extended conformation, which is entangled around the misfolded protein, is the major conformation forming aggregates. At high temperature, native-like is the major conformation forming in aggregates at a dilute protein concentration while the extended one becomes dominant at high concentration. Folding kinetics described by (kc) and (kf), representing the collapsing and folding kinetic parameters, respectively, are also found to be determined by protein concentration and temperature. The increase in protein concentration leads to a monotonous decrease of kc while a bell shape response of kf, indicating the existence of an optimum concentration at which the intermolecular force between proteins accelerates protein folding. On the other hand, the over-intensive interaction between proteins hinders protein folding via forming irreversible aggregates. At all protein concentrations, an optimal collapsing and folding temperature exist. The increase in protein concentration and temperature leads to the acceleration of protein aggregation, making the aggregating kinetics over the folding. T=150K and C=0.6152mM are found to be the optimal folding condition, at which conformations with native contact (NC) > 30 are dominant within 2ns, favoring formation of native conformation. The aggregating contact (AGC) is minor when t<1ns and only slightly increases within 10ns. At T=300K and C=0.6152mM, however, protein aggregation occurs and continues throughout the folding process. At high protein concentration, aggregation occurs during the whole process with low fraction of native conformation, indicating the over-intensive interaction between proteins hinders protein folding. A temperature-shift strategy in terms of low temperature ? high temperature ? low temperature was applied to protein folding at C=0.6152mM, C=1.23mM and C=3.076mM, respectively. The folding yield was 81%, 72% and 51%, while the control one operated at the optimal constant temperature was 67%, 62% and 42%, respectively. Above simulation gives molecular insight of protein folding and aggregation and is helpful for the design of suitable folding strategy for the production of recombinant protein from inclusion bodies.