Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins

Abstract

Structural information regarding metallothioneins (MTs) has been hard to come by due to its highly dynamic nature in the absence of metal-thiolate cluster formation and crystallization difficulties. Thus, typical spectroscopic methods for structural determination are limited in their usefulness when applied to MTs. Mass spectrometric methods have revolutionized our understanding of protein dynamics, structure, and folding. Recently, advances have been made in residue modification mass spectrometry in order to probe the hard-to-characterize structure of apo- and partially metalated MTs. By using different cysteine specific alkylation reagents, time dependent electrospray ionization mass spectrometry (ESI-MS), and step-wise “snapshot” ESI-MS, we are beginning to understand the dynamics of the conformers of apo-MT and related species. In this review we highlight recent papers that use these and similar techniques for structure elucidation and attempt to explain in a concise manner the data interpretations of these complex methods. We expect increasing resolution in our picture of the structural conformations of metal-free MTs as these techniques are more widely adopted and combined with other promising tools for structural elucidation.

Metallothionein: An Aggressive Scavenger—The Metabolism of Rhodium(II) Tetraacetate (Rh2(CH3CO2)4)

“…Structural characteristics can be inferred from the reaction profile of modification observed in the mass spectral data. 106 As expected, the higher metalated Rh 4 and Rh 6 -bound MT shows less solvent accessibility because of the lower spread of the p (BQ)-modified species. The most solvent accessible species, Rh 2 MT, shows two distinct distributions, indicating that two metalated states are created.…”

“…Similarly to Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid)), this irreversible binding permits quantification of free thiols. 37,92,106−108 This is observed as a predictable mass change, but in addition with respect to MT and its 20 cysteines, the modified speciation distribution can also provide information about the solvent accessibility of the cysteines. 108 This technique has been used to understand metalation of apo-βαMT through partially metalated and fully metalated Cu 6 MT, Cu 13 MT, Cu 20 MT, and As 6 MT.…”

“…As described by Irvine and Stillman, cysteine accessibility can be understood by the distribution of cysteine modified species. 106 A normal distribution represents statistically equal accessibility of the modifier to all available free cysteines. This means that the unbound cysteines are exposed.…”

Collision-Induced Unfolding of Partially Metalated Metallothionein-2A: Tracking Unfolding Reactions of Gas-Phase Ions

“…Fenselau et al pioneered the use of electrospray ionization mass spectrometry (ESI-MS) for studies of metallothioneins, and Stillman et al have championed ESI for studies of ion reactivity and kinetics of metalation reactions. − More recently, we have made extensive use of nanoelectrospray ionization mass spectrometry in combination with ion mobility (IM) spectrometry (nESI-IM-MS) to determine the conformational preferences of both partially and fully metalated MTs. − IM-MS has been applied to protein structure study and can be used to probe protein dynamics and shows great potential for structural characterization of natively unfolded MTs, specifically conformational changes upon binding of metal ions or other folding effectors. − As examples, Chen et al combined bottom-up and top-down nESI-IM-MS proteomic approaches with chemical labeling to unambiguously show that metalation involves sequential addition of Cd 2+ to the α domain resulting in preferential formation of an intermediate, Cd 4 -MT2, but at higher metal ion concentrations, the excess Cd 2+ ions add to the β domain to yield the fully metalated Cd 7 -MT2 . Likewise, demetalation (by EDTA) occurs in the reverse order .…”

The content of metals in metallothioneins of the bivalve mollusk Unio tumidus depen­ding on different in situ and in vitro exposures

“…The most important example is connecting to the detoxification of Cd, whereas MTs can bind almost all cadmium content within the cells in complexes of low toxi city [11]. Due to this unique property, MTs are considered to be an ideal tool for bioremediation of toxic metal contaminated environments [4,8]. However, utilizing of the MTs with the purpose of bioremediation is discussed only for the bacteria [4].…”