The folding of many small proteins is kinetically a two-state process with one major free-energy barrier to overcome,which can be roughly regarded as the inverse process of unfolding.In this work,we first use a Gaussian network model to predict the folding nucleus corresponding to the major free-energy barrier of protein 2GB1,and find that the folding nucleus is located in the β-sheet domain.High-temperature molecular dynamics simulations are then used to investigate the unfolding process of 2GB1.We draw free-energy surface from unfolding simulations,taking RMSD and contact number as reaction coordinates,which confirms that the folding of 2GBl is kinetically a two-state process.The comparison of the contact maps before and after the free energy barrier indicates that the transition from native to non-native structure of the protein is kinetically caused by the destruction of the β-sheet domain,which manifests that the folding nucleus is indeed located in the(-sheet domain.Moreover,the constrained MD simulation further confirms that the destruction of the secondary structures does not alter the topology of the protein retained by the folding nucleus.These results provide vital information for upcoming researchers to further understand protein folding in similar systems.