Retro operation on the Trp-cage miniprotein sequence produces an unstructured molecule capable of folding similar to the original only upon 2,2,2-trifluoroethanol addition.

Amino acid sequence and environment are the most important factors determining the structure, stability and dynamics of proteins. To evaluate their roles in the process of folding, we studied a retroversion of the well-described Trp-cage miniprotein in water and 2,2,2-trifluoroethanol (TFE) solution. We show, by circular dichroism spectroscopy and nuclear magnetic resonance (NMR) measurement, that the molecule has no stable structure under conditions in which the Trp-cage is folded. A detectable stable structure of the retro Trp-cage, with the architecture similar to that of the original Trp-cage, is established only upon addition of TFE to 30% of the total solvent volume. The retro Trp-cage structure shows a completely different pattern of stabilizing contacts between amino acid residues, involving the guanidinium group of arginine and the aromatic group of tryptophan. The commonly used online prediction methods for protein and peptide structures Robetta and PEP-FOLD failed to predict that the retro Trp-cage is unstructured under default prediction conditions. On the other hand, both methods provided structures with a fold similar to those of the experimentally determined NMR structure in water/TFE but with different contacts between amino acids.

Enantioselective effects of alpha-hexachlorocyclohexane (HCH) isomers on androgen receptor activity in vitro.

Alpha-hexachlorocyclohexane (alpha-HCH), a part of the HCH pesticide mixture, is one of the most widespread persistent organic pollutants. Interestingly, only limited number of studies addressed the toxicity of alpha-HCH and the effects of its individual optical isomers have not been investigated in detail. In the present study we separated two alpha-HCH enantiomers by preparative HPLC and studied their activities towards androgen receptor (AR) using the MDA-kb2 cell line stably transfected with the luciferase reporter gene under the control of AR. There was no direct effect of alpha-HCH on AR but both isomers significantly suppressed the activity of AR in co-exposure with the natural ligand dihydrotestosterone in a concentration-dependent manner. One of the enantiomers appeared to be more active at lower concentration, which was also supported by the molecular modeling calculations with AR that showed a slight difference in estimated free energy of binding and inhibition constant between two enantiomers. Although studies with other pesticides demonstrated strong enantioselective differences in toxicity, the present research shows rather minor differences in modulations of AR by both alpha-HCH enantiomers. For the first time, enantioselective effects of alpha-HCH were demonstrated and the results suggest interaction with multiple regulatory events controlling the AR activity. Full elucidation of the toxicity mechanism will require further research.