An optimal Mg2+ concentration for kinetic folding of the Tetrahymena ribozyme

Divalent metal ions, such as Mg2+, are generally required for tertiary structure formation in RNA. Although the role of Mg2+ binding in RNA-folding equilibria has been studied extensively, little is known about the role of Mg2+ in RNA-folding kinetics. In this paper, we explore the effect of Mg2+ on the rate-limiting step in the kinetic folding pathway of the Tetrahymena ribozyme. Analysis of these data reveals the presence of a Mg2+-stabilized kinetic trap that slows folding at higher Mg2+ concentrations. Thus, the Tetrahymena ribozyme folds with an optimal rate at 2 mM Mg2+, just above the concentration required for stable structure formation. These results suggest that thermodynamic and kinetic folding of RNA are cooptimized at a Mg2+ concentration that is sufficient to stabilize the folded form but low enough to avoid kinetic traps and misfolding.

Design of potent and selective human cathepsin K inhibitors that span the active site

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.