Protein Expr Purif. 2009 Jul 3; Guo C, Lian Y, Liu Q, Liu J, Zhang Y, Lin DMouse lipocalin6 (mLcn6) was recently identified to be specifically expressed in the epididymis and speculated to may play a role in sperm maturation. However, further studies were hindered due to the bottleneck to obtain enough recombinant mLcn6 proteins. In this article, GB1 tag was successfully applied to improve the soluble expression of mLcn6. Thermal unfolding experiments demonstrate that GB1 can enhance the structural stability of mLcn6. Fluorescence spectroscopy experiments show that mLcn6 prepared according to our procedure has high affinities to both retinoic acid (K(d) = 810 nM) and retinol (K(d) = 210 nM). In conclusion, soluble, stable and active mLcn6 was recombinantly prepared with the help of the GB1 tag, which will facilitate the structural and functional studies of mLcn6.

J Phys Chem B. 2009 Jul 7; Shen J, Li W, Liu G, Tang Y, Jiang HEstrogen receptors (ER) belong to the nuclear receptor superfamily, and two subtypes, ERalpha and ERbeta, have been identified to date. The differentiated functions and receptor expressions of ERalpha and ERbeta made it attracted to discover subtype-specified ligands with high selectivity. However, these two subtypes are highly homologous and only two residues differ in the ligand binding pocket. Therefore, the mechanism of ligand selectivity has become an important issue in searching selective ligands of ER subtypes. In this study, steered molecular dynamics simulations were carried out to investigate the unbinding pathways of two selective ERbeta ligands from the binding pocket of both ERalpha and ERbeta, which demonstrated that the pathway between the H11 helix and the H7 approximately H8 loop was the most probable for ligand escaping. Then potentials of mean force for ligands unbinding along this pathway were calculated in order to gain insights into the molecular basis for energetics of ligand unbinding and find clues of ligand selectivity. The results indicated that His524/475 in ERalpha/ERbeta acted as a "gatekeeper" during the ligand unbinding. Especially, the H7 approximately H8 loop of ERbeta acted as a polar "transmitter" that controlled the ligand unbinding from the binding site and contributed to the ligand selectivity. Finally, the mechanism of ligand selectivity of ER subtypes was discussed from a kinetic perspective and suggestions for improving the ligand selectivity of ERbeta were also presented. These findings could be helpful for rational design of highly selective ERbeta ligands.