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Hydrogen bonds and their relative strengths in proteins are of importance for understanding protein structure and protein motions. The correct strength of such hydrogen bonds is experimentally known to vary greatly from Ϸ5-6 kcal͞mol for the isolated bond to Ϸ0.5-1.5 kcal͞mol for proteins in solution. To estimate these bond strengths, here we suggest a direct novel kinetic procedure. This analyzes the timing of the trajectories of a properly averaged dynamic ensemble. Here we study the observed rupture of these hydrogen bonds in a molecular dynamics calculation as an alternative to using thermodynamics. This calculation is performed for the isolated system and contrasted with results for water. We find that the activation energy for the rupture of the hydrogen bond in a ␤-sheet under isolated conditions is 4.76 kcal͞mol, and the activation energy is 1.58 kcal͞mol for the same ␤-sheet in water. These results are in excellent agreement with observations and suggest that such a direct calculation can be useful for the prediction of hydrogen bond strengths in various environments of interest.T he strength of the hydrogen bond in the linking of protein structures particular in a water environment is of essential importance to predict the activity of proteins such as enzyme action, protein folding, binding of proteins, and many other processes (1, 2). Although much has been written on protein dynamics in water (3), a detailed energy calculation including the correct water environment has been difficult to put into a computational framework. The energetics of hydrogen bonds within proteins is known to undergo large changes in water. The effect of water is also process dependent, so it is different here from protein signal transport (4). Such environmental changes in a hydrogen bond strength are important to the understanding of protein interactions, including drug design (5, 6). The drugreceptor hydrogen bond is operative in many applications (7).Hydrogen bonds are one of the major structural determinants, controlling active configurations by connecting protein structure in a fluxional equilibrium. The making and breaking of hydrogen bonds profoundly affects the rates and dynamic equilibria, which are responsible for much of the biological activity of proteins. This behavior is strongly medium dependent, so the action of these hydrogen bonds in isolated systems is quite different from the action in a water environment. The complex environment presented to the hydrogen bond by water is not easy to incorporate in calculations, but it is of major relevance, and results obtained need to be checked against experiments. A huge and complex phase space contributes to the effects of entropy on the hydrogen bond, particularly in water, and thus influences the free energy of these bonds. The general task of assessing the entropic contributions to the dynamic strength of these bonds is a matter of extensive research (8) and is difficult to quantify. Furthermore, it must be recognized that various relevant bonding environments will...

BackgroundIn osteoarthritis (OA), the imbalance of chondrocytes’ anabolic and catabolic factors can induce cartilage destruction. Interleukin-1 beta (IL-1β) is a potent pro-inflammatory cytokine that is capable of inducing chondrocytes and synovial cells to synthesize MMPs. The hypoxia-inducible factor-2alpha (HIF-2alpha, encoded by Epas1) is the catabolic transcription factor in the osteoarthritic process. The purpose of this study is to validate the effects of ecdysteroids (Ecd) on IL-1β- induced cartilage catabolism and the possible role of Ecd in treatment or prevention of early OA.MethodsChondrocytes and articular cartilage was harvested from newborn ICR mice. Ecd effect on chondrocytes viability was tested and the optimal concentration was determined by MTT assay. The effect of HIF-2α (EPAS1) in cartilage catabolism simulated by IL-1β (5 ng/ml) was evaluated by articular cartilage explants culture. The effects of Ecd on IL-1β-induced inflammatory conditions and their related catabolic genes expression were analyzed.ResultsInterleukin-1β (IL-1β) treatment on primary mouse articular cartilage explants enhanced their Epas1, matrix metalloproteinases (MMP-3, MMP-13) and ADAMTS-5 genes expression and down-regulated collagen type II (Col2a1) gene expression. With the pre-treatment of 10−8M Ecd, the catabolic effects of IL-1β on articular cartilage were scavenged.ConclusionIn conclusions, Ecd can reduce the IL-1β-induced inflammatory effect of the cartilage. Ecd may suppress IL-1β- induced cartilage catabolism via HIF-2α pathway.

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