Protein-Ligand Affinity Determinations Using Covalent Labeling-Mass Spectrometry.

Determining the binding affinity is an important aspect of characterizing protein-ligand complexes. Here, we describe an approach based on covalent labeling (CL)-mass spectrometry (MS) that can accurately provide protein-ligand dissociation constants ( values) using diethylpyrocarbonate (DEPC) as the labeling reagent. Even though DEPC labeling reactions occur on a time scale that is similar to the dissociation/reassociation rates of many protein-ligand complexes, we demonstrate that relatively accurate binding constants can still be obtained as long as the extent of protein labeling is kept below 30%.

Epigallocatechin-3-gallate Inhibits Cu(II)-Induced β-2-Microglobulin Amyloid Formation by Binding to the Edge of Its β-Sheets.

Epigallocatechin-3-gallate (EGCG) is a catechin found in green tea that can inhibit the amyloid formation of a wide variety of proteins. EGCG's ability to prevent or redirect the amyloid formation of so many proteins may reflect a common mechanism of action, and thus, greater molecular-level insight into how it exerts its effect could have broad implications. Here, we investigate the molecular details of EGCG's inhibition of the protein β-2-microglobulin (β2m), which forms amyloids in patients undergoing long-term dialysis treatment.

Structural Heterogeneity in the Preamyloid Oligomers of β-2-Microglobulin.

In dialysis patients, the protein β2-microglobulin (β2m) forms amyloid fibrils in a condition known as dialysis-related amyloidosis. To understand the early stages of the amyloid assembly process, we have used native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble preamyloid oligomers. ESI-IM-MS reveals the presence of multiple conformers for the dimer, tetramer, and hexamer that precede the Cu(II)-induced amyloid assembly process, results which are distinct from β2m oligomers formed at low pH.

Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules.

Amyloid aggregates are associated with several debilitating diseases, and there are numerous efforts to develop small molecule treatments against these diseases. One challenge associated with these efforts is determining protein binding site information for potential therapeutics because amyloid-forming proteins rapidly form oligomers and aggregates, making traditional protein structural analysis techniques challenging. Using β-2-microglobulin (β2m) as a model amyloid-forming protein along with two recently identified small molecule amyloid inhibitors (i.

Small molecule-mediated inhibition of β-2-microglobulin-based amyloid fibril formation.

In dialysis patients, β-2 microglobulin (β2m) can aggregate and eventually form amyloid fibrils in a condition known as dialysis-related amyloidosis, which deleteriously affects joint and bone function. Recently, several small molecules have been identified as potential inhibitors of β2m amyloid formation Here we investigated whether these molecules are more broadly applicable inhibitors of β2m amyloid formation by studying their effect on Cu(II)-induced β2m amyloid formation. Using a variety of biophysical techniques, we also examined their inhibitory mechanisms.