Specific interactions and binding free energies between thermolysin and dipeptides: molecular simulations combined with ab initio molecular orbital and classical vibrational analysis.

Thermolysin (TLN) is a metalloprotease widely used as a nonspecific protease for sequencing peptide and synthesizing many useful chemical compounds by the chemical industry. It was experimentally shown that the activity and functions of TLN are inhibited by the binding of many types of amino acid dipeptides. However, the binding mechanisms between TLN and dipeptides have not been clarified at the atomic and electronic levels. In this study, we investigated the binding mechanisms between TLN and four dipeptides. Specific interactions and binding free energies (BFEs) between TLN and the dipeptides were calculated using molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital (FMO) methods. The molecular systems were embedded in solvating water molecules during calculations. The calculated BFEs were qualitatively consistent with the trend of the experimentally observed inhibition of TLN activity by binding of the dipeptides. In addition, the specific interactions between the dipeptides and each amino acid residue of TLN or solvating water molecules were elucidated by the FMO calculations.

Effects of Dietary Brown Rice on the Growth Performance, Systemic Oxidative Status, and Splenic Inflammatory Responses of Broiler Chickens under Chronic Heat Stress.

The aim of this study was to evaluate the effects of dietary brown rice on the growth performance, systemic oxidative status, and splenic inflammatory responses of broiler chickens under both thermo-neutral and chronic heat stress conditions. Forty 12-day-old male broiler chickens (ROSS 308) were randomly assigned to two groups and fed either a control diet (corn-based) or a brown rice-based diet. After seven days (19 days old), both groups were randomly divided into two sub-groups (n=10), one of which was exposed to heat stress (33°C for 14 days), while the other was maintained at 24°C. Heat exposure reduced the body weight gain and feed intake (p<0.01) of both groups. In terms of oxidative plasma states, heat exposure reduced the glutathione peroxidase activity and increased the ceruloplasmin content, while the 2-thiobarbituric acid reactive substance and reduced glutathione levels were not affected adversely. Heat exposure activated the immune responses, as evidenced by increased plasma immunoglobin levels, and altered splenic immune-related gene expressions including heat shock proteins, toll-like receptor 4, and interleukin-12. Under both thermo-neutral and heat stress conditions, dietary brown rice improved the growth performance, decreased the immunoglobulin levels, and down-regulated the expression of splenic immune-related genes of broilers, although their systemic oxidative status was not affected. Dietary brown rice should be considered as a valuable component of broiler chicken feeds subjected to both thermo-neutral and heat stress conditions. The positive effects of brown rice on bird performance may be associated with the modulation of the immune responses, as reflected by the decreased production of immunoglobulins and altered splenic immune-related gene expression.

Specific interactions and binding energies between thermolysin and potent inhibitors: molecular simulations based on ab initio molecular orbital method.

Biochemical functions of the metalloprotease thermolysin (TLN) are controlled by various inhibitors. In a recent study we identified 12 compounds as TLN inhibitors by virtual screening and in vitro competitive binding assays. However, the specific interactions between TLN and these inhibitors have not been clarified. We here investigate stable structures of the solvated TLN-inhibitor complexes by classical molecular mechanics simulations and elucidate the specific interactions between TLN and these inhibitors at an electronic level by using ab initio fragment molecular orbital (FMO) calculations. The calculated binding energies between TLN and the inhibitors are qualitatively consistent with the experimental results, and the FMO results elucidate important amino acid residues of TLN for inhibitor binding. Based on the calculated results, we propose a novel potent inhibitor having a large binding affinity to TLN.