REMD and umbrella sampling simulations to probe the energy barrier of the folding pathways of engrailed homeodomain.

Proteins fold by diverse pathways which depend on the energy barriers involved in reaching different intermediates. There has been a lot of development in the theoretical aspects of protein folding, from force-field to simulation techniques. One such simulation approach is replica exchange molecular dynamics simulation (REMD), which provides an efficient conformational sampling method to understand the events involved in protein folding. In this study, an attempt is made to explore the folding funnel of engrailed homeodomain protein (EnHD) using REMD simulations. EnHD is a 54 residue long helix bundle protein which has a folding time of about 15 μs. The protein was represented using the Amber United atom model in order to reduce the system size which helped to speed up the simulation. Individual replicas were simulated for 1.4-2 μs making cumulative time of more than 100 μs of REMD simulations. Free energy analysis was carried out to understand the folding behavior of EnHD protein. Effects of temperature range and exchange frequency in REMD simulations have been explored. In addition to this, multiple umbrella sampling (US) simulations of a total of 320 ns were also carried out, followed by weighted histogram analysis method (WHAM) to investigate the energy barriers involved during the folding of various intermediates. US studies were also carried on mutational variants of EnHD protein to see effect of the mutations on the folding pathway of the protein. The use of US technique may be helpful for predicting fast folding mutants or protein engineering. The combination of REMD with US may help in understanding the energetics between multiple pathways of fast folding proteins and their mutant counterparts.