Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.

Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation.

Mapping inhibitor binding modes on an active cysteine protease via nuclear magnetic resonance spectroscopy.

Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping.

Inhibition of a viral enzyme by a small-molecule dimer disruptor.

We identified small-molecule dimer disruptors that inhibit an essential dimeric protease of human Kaposi's sarcoma-associated herpesvirus (KSHV) by screening an alpha-helical mimetic library. Next, we synthesized a second generation of low-micromolar inhibitors with improved potency and solubility. Complementary methods including size exclusion chromatography and 1H-13C HSQC titration using selectively labeled 13C-Met samples revealed that monomeric protease is enriched in the presence of inhibitor.

Substrate modulation of enzyme activity in the herpesvirus protease family.

The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme.

Dissection of RAP-LRP interactions: binding of RAP and RAP fragments to complement-like repeats 7 and 8 from ligand binding cluster II of LRP.

The receptor associated protein (RAP) is a three domain 38kDa ER-resident chaperone that helps folding of LRP and other LDL receptor family members and prevents premature binding of protein ligands. It competes strongly with all known LRP ligands. To further understanding of the specificity of RAP-LRP interactions, the binding of RAP and RAP fragments to two domains (CR7-CR8) from one of the main ligand-binding regions of LRP has been examined by 2D HSQC NMR spectroscopy and isothermal titration calorimetry.

Structural organization of the receptor associated protein.

The receptor associated protein (RAP) is a 38 kDa ER-resident protein that binds tightly to the low density lipoprotein receptor-related protein (LRP), and other members of the LDL receptor family of receptors, and competes with all known LRP ligands for binding to LRP. To better understand the domain structure and organization of RAP, we have expressed RAP subfragments and examined them by two-dimensional HSQC NMR and fluorescence spectroscopies, by differential scanning calorimetry, and by both equilibrium and velocity sedimentation measurements. We found that the protein is organized into three domains located in the first third (1D), middle third (2D), and last third (3D) of the protein.