Occupying a flat subpocket in a tRNA-modifying enzyme with ordered or disordered side chains: Favorable or unfavorable for binding?

Small-molecule ligands binding with partial disorder or enhanced residual mobility are usually assumed as unfavorable with respect to their binding properties. Considering thermodynamics, disorder or residual mobility is entropically favorable and contributes to the Gibbs energy of binding. In the present study, we analyzed a series of congeneric ligands inhibiting the tRNA-modifying enzyme TGT.

Beyond affinity: enthalpy-entropy factorization unravels complexity of a flat structure-activity relationship for inhibition of a tRNA-modifying enzyme.

Lead optimization focuses on binding-affinity improvement. If a flat structure-activity relationship is detected, usually optimization strategies are abolished as unattractive. Nonetheless, as affinity is composed of an enthalpic and entropic contribution, factorization of both can unravel the complexity of a flat, on first sight tedious SAR.

Chasing protons: how isothermal titration calorimetry, mutagenesis, and pKa calculations trace the locus of charge in ligand binding to a tRNA-binding enzyme.

Drug molecules should remain uncharged while traveling through the body and crossing membranes and should only adopt charged state upon protein binding, particularly if charge-assisted interactions can be established in deeply buried binding pockets. Such strategy requires careful pKa design and methods to elucidate whether and where protonation-state changes occur. We investigated the protonation inventory in a series of lin-benzoguanines binding to tRNA-guanine transglycosylase, showing pronounced buffer dependency during ITC measurements.

Impact of protein and ligand impurities on ITC-derived protein-ligand thermodynamics.

Background: The thermodynamic characterization of protein-ligand interactions by isothermal titration calorimetry (ITC) is a powerful tool in drug design, giving valuable insight into the interaction driving forces. ITC is thought to require protein and ligand solutions of high quality, meaning both the absence of contaminants as well as accurately determined concentrations.

Methods: Ligands synthesized to deviating purity and protein of different pureness were titrated by ITC.

Structural analysis of coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum reveals a novel active-site environment.

Coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum (Linaceae) catalyzes the unusual methylation of the side-chain hydroxyl group of coniferyl alcohol. The protein was heterologously expressed in Escherichia coli as a hexahistidine derivative and purified for crystallization. Diffracting crystals were obtained of the pure protein and of its selenomethionine derivative, as well as of complexes with coniferyl alcohol and with S-adenosyl-L-homocysteine together with coniferyl alcohol 9-O-methyl ether (PDB entries 4ems, 4e70 and 4evi, respectively).

Targeting the blind spot of polycationic nanocarrier-based siRNA delivery.

Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA vs pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery.