Relationship of Thermostability and Binding Affinity in Metal-binding WW-Domain Minireceptors.

The design of metallo-miniproteins advances our understanding of the structural and functional roles of metals in proteins. We recently designed a metal-binding WW domain, WW-CA-Nle, which displays three histidine residues on its surface for coordination of divalent metals Ni(II), Zn(II) and Cu(II). However, WW-CA-Nle is a molten globule in the apo state and thus showed only moderate binding affinities with Kd values in the µM regime. In this report, we hypothesize that improved thermal stability of the apo state of the metal binding WW-domain scaffold should lead to improved preorganization of the metal-binding site and consequently to higher metal-binding affinities. By redesigning WW-CA-Nle, we obtained WW-CA variants, WW-CA-min and WW-CA-ANG, which were fully folded in the apo states and displayed moderate to excellent thermostabilities in the apo and holo states. We were able to show that the improved thermal stabilities led to improved metal binding, which was reflected in Kd values that were at least one order of magnitude lower compared to WW-CA-Nle. EPR spectroscopy and ITC measurements revealed a better defined and predisposed metal binding site in WW-CA-ANG.

Synthesis of New Steroidal Carbamates with Plant-Growth-Promoting Activity: Theoretical and Experimental Evidence.

A priority of modern agriculture is to use novel and environmentally friendly plant-growth promoter compounds to increase crop yields and avoid the indiscriminate use of synthetic fertilizers. Brassinosteroids are directly involved in the growth and development of plants and are considered attractive candidates to solve this problem. Obtaining these metabolites from their natural sources is expensive and cumbersome since they occur in extremely low concentrations in plants. For this reason, much effort has been dedicated in the last decades to synthesize brassinosteroids analogs. In this manuscript, we present the synthesis and characterization of seven steroidal carbamates starting from stigmasterol, ß-sitosterol, diosgenin and several oxygenated derivatives of it. The synthesis route for functionalization of diosgenin included epoxidation and epoxy opening reactions, reduction of carbonyl groups, selective oxidation of hydroxyl groups, among others. All the obtained compounds were characterized by 1H and 13C NMR, HRMS, and their melting points are also reported. Rice lamina inclination test performed at different concentrations established that all reported steroidal carbamates show plant-growth-promoting activity. A molecular docking study evaluated the affinity of the synthesized compounds towards the BRI1-BAK1 receptor from Arabidopsis thaliana and three of the docked compounds displayed a binding energy lower than brassinolide.