Chromatographic separation of similar post-translationally modified metallothioneins reveals the changing conformations of apo-MT upon cysteine alkylation by high resolution LC-ESI-MS

Publication date: Available online 5 March 2018 Source:Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics Author(s): Gordon W. Irvine, Martin J. Stillman Metallothioneins (MTs) are a class of small cysteine-rich proteins essential for Zn and Cu homeostasis, heavy metal detoxification, and cellular redox chemistry. Herein, we describe the separation and characterization of MTs differentially modified with N-ethylmaleimide (NEM) by liquid chromatography-mass spectrometry (LC-MS). The full-length recombinant MT isoform 1a as well as is isolated domain fragments were first alkylated, then separated on column with subsequent detection by MS. Different behavior was observed for the three peptides with the full-length protein and the isolated α-domain exhibiting similar separation characteristics. For the isolated β-domain, the smallest peptide, each alkylated species was well separated, indicating large changes in protein conformation. For the full-length and α-domain peptides, the apo- and lightly alkylated species co-eluted, indicating similar structural properties. However, the more extensively alkylated species were well separated from each other, indicating the sequential unfolding of the apo-MT peptides and providing evidence for the mechanistic explanation for the cooperative alkylation reaction observed for NEM and other bulky and hydrophobic alkylation reagents. We show for the first time clear separation of highly similar MTs, differing by only +125 Da...

Source: Biochimica et Biophysica Acta (BBA) Proteins and Proteomics - March 6, 2018 Category: Biochemistry Source Type: research