Selective cysteine modification of metal‐free human metallothionein 1a and its isolated domain fragments: Solution structural properties revealed via ESI‐MS

Abstract

Human metallothionein 1a, a protein with two cysteine-rich metal-binding domains (α with 11 Cys and β with 9), was analyzed in its metal-free form by selective, covalent Cys modification coupled with ESI-MS. The modification profiles of the isolated β- and α-fragments reacted with p-benzoquinone (Bq), N-ethylmalemide (NEM) and iodoacetamide (IAM) were compared with the full length protein using ESI-mass spectral data to follow the reaction pathway. Under denaturing conditions at low pH, the reaction profile with each modifier followed pathways that resulted in stochastic, Normal distributions of species whose maxima was equal to the mol. eq. of modifier added. Our interpretation of modification at this pH is that reaction with the cysteines is unimpeded when the full protein or those of its isolated domains are denatured. At neutral pH, where the protein is expected to be folded in a more compact structure, there is a difference in the larger Bq and NEM modification, whose reaction profiles indicate a cooperative pattern. The reaction profile with IAM under native conditions follows a similar stochastic distribution as at low pH, suggesting that this modifier is small enough to access the cysteines unimpeded by the compact structure. The data emphasize the utility of residue modification coupled with electrospray ionization mass spectrometry for the study of protein structure.

Formation of oxidative and non-oxidative dimers in metallothioneins: Implications for charge-state analysis for structural determination

“…This indicates that oxidative dimer formation is not likely, even in the apo‐protein with many redox active Cys residues, as long as precautions are taken to limit oxygen exposure. This fits with structural models of apo‐MTs that have proposed the Cys residues to be buried within the protein as a globular structure, similar to the holoprotein …”

“…In the unfolded metal‐free state, the charge‐state distribution shifts to a slightly higher range, as can be seen in this and other works. When all Cys residues in MT are covalently modified by N ‐ethylmalemide, adding 2500 Da to the mass of the protein, the average charge‐state shift is increased by less than 2 . A report by our group showed a large shift in the charge states of the apo‐protein upon acidification to pH 1.8, in contrast to most other studies which show only a slight shift in charge states .…”

Proteomic High Affinity Zn2+ Trafficking: Where Does Metallothionein Fit in?

“…That the apo-protein adopts a secondary structure is supported by experiments showing that the reactivity of cysteinyl sulfhydryl groups differs in native and denatured apo-MT, and that the rate of binding of Cd 2+ to the protein also varies between these two states [95,96]. …”

Isolated domains of recombinant human apo-metallothionein 1A are folded at neutral pH: a denaturant and heat-induced unfolding study using ESI-MS

“…That the relative abundance can be extracted with confidence from the ESI-MS experiment indicates that the structural changes upon metalation do not result in radically different ionization efficiencies between species. Conclusion from these many reported methods, including techniques such as charge state analysis [ 19 ], ion-mobility (IM) MS [ 20 ], and residue modification coupled with ESI-MS [ 21 ], and LC-MS [ 22 ], cast doubt on the model of a completely unstructured apo-MT at physiological pH. However, these mass spectral studies were not the first to suggest a compact and globular structure, as theoretical calculations predicted that the cysteine residues in the apo-MTs were buried and not freely accessible to the solvent [ 23 ] and FRET studies [ 24 , 25 ] of recombinant MTs in the early 2000s showed that there was also negligible volume change in the protein upon metalation.…”

“…Recently, our group and others have demonstrated that the reaction with cysteine alkylation agents, such as n -ethylmaleimide (NEM), p -benzoquinone, and iodoacetamide can be used to determine the folded state of apo-MT under various conditions [ 21 ]. The most common denaturants used in biochemical unfolding studies are acidity and guanidinium chloride (GdmCl).…”