Combining Chemical Labeling, Bottom-Up and Top-Down Ion-Mobility Mass Spectrometry To Identify Metal-Binding Sites of Partially Metalated Metallothionein

Chen, Shu Hua; Russell, William K.; Russell, David H.

doi:10.1021/ac303522h

Cited by 42 publications

(107 citation statements)

References 69 publications

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“…Interestingly, those residues are exposed to the solvent in the isolated b-domain, but are buried in the whole protein MT2. 67 Our molecular dynamics indicated that Cys5 and Cys29 are free of coordination (Fig. S14c, ESI †) in which the first one interacts with Gln23 via H-bonding (2.6 Å).…”

Section: Zn(ii)-dependent Folding Of the Isolated B-domain Differs Frmentioning

confidence: 92%

“…25,87,88 On the other hand, corresponding ESI-MS titration studies showed that intermediate forms (Cd 1-3 MT2) are also formed; however, Cd 4 MT2 is highly favourable. 29,67 It is worth noting that exact identification of successive Cd(II) binding events and sites in the a-domain has not been performed to date and such studies could possibly reveal differences or the lack of differences in Cd(II) affinities toward the a-domain. Data collected to date show that both metal ions bind to the a-domain specifically with high affinity, but Zn(II) binding is more sequential.…”

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confidence: 99%

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Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

et al. 2018

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a Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M 4 S 11 (a) and M 3 S 9 (b) clusters. Recent discoveries indicate that Zn(II) ions are bound with MT2 with the range from nano-to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(II)-depleted MT2 species. These forms serve as Zn(II) donors or acceptors and are formed under varying cellular free Zn(II) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(II) binding mechanism in human MT2 with high resolution with respect to particular Zn(II) binding sites, and provide structural insights into Zn(II)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the a-domain is formed sequentially in the first stages, followed by the formation of the b-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS 4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the b-domain upon applying the seventh Zn(II) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(II) ion associates with the b-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(II) binding to the isolated b-domain differs significantly from the whole protein, which explains its previously observed different Zn(II)-binding properties. MD results obtained for Zn(II) binding to the whole protein and isolated b-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis. Significance to metallomicsThe growing interest in metallomics within metal-dependent biological systems provides an opportunity to explore the structural and buffering properties of metallothioneins (MTs). The wide relevance of MTs is obvious due to their multiple biological functions such as participation in zinc and copper homeostasis. However, the MT metalloproteome is still not fully explored and is rather complicated in vivo or in vitro because of its highly dynamic structure, which lacks secondary structural elements and metamorphic behaviour. We have undertaken compreh...

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Section: Zn(ii)-dependent Folding Of the Isolated B-domain Differs Frmentioning

confidence: 92%

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confidence: 99%

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

et al. 2018

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“…ApoMT sample (Bestenbalt LLC, Tallinn, Estonia) was reconstituted to 7 μM using 50 mM ammonium acetate (pH 7.4) solution containing 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl; Thermo Fisher Scientific, Rockford, IL, USA) [29,42]. Metalated Cd i MT (i = 1 − 7) were obtained by sequential addition of 1, 2, 3, 4, 5, 6, and 7 μL of 1 mM of Cd 2+ to 100 μL of a 7 μM apoMT solution.…”

Section: Sample Preparationmentioning

confidence: 99%

“…Metalated Cd i MT (i = 1 − 7) were obtained by sequential addition of 1, 2, 3, 4, 5, 6, and 7 μL of 1 mM of Cd 2+ to 100 μL of a 7 μM apoMT solution. Care was taken in all ESI-IM-MS experiments reported here to exclude ESI-induced protein oxidation and nonspecific metal binding as previously described [29,42].…”

Section: Sample Preparationmentioning

confidence: 99%

How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?

Chen

Russell

2015

J. Am. Soc. Mass Spectrom.

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143

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by "native-state" ESI show a strong dependence on E int . Collisional activation is used to increase E int prior to the ions entering and within the traveling wave (TW) ion mobility analyzer. Comparisons of experimental CCSs with those generated by molecular dynamics (MD) simulation for solution-phase ions and solvent-free ions as a function of temperature provide new insights about conformational preferences and retention of solution conformations. The E int -dependent CCSs, which reveal increased conformational diversity of the ion population, are discussed in terms of folding/unfolding of solvent-free ions. For example, ubiquitin ions that have low internal energies retain native-like conformations, whereas ions that are heated by collisional activation possess higher internal energies and yield a broader range of CCS owing to increased conformational diversity due to losses of secondary and tertiary structures. In contrast, the CCS profile for the IDP apoMT is consistent with kinetic trapping of an ion population composed of a wide range of conformers, and as the E int is increased, these structurally labile conformers unfold to an elongated conformation.

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“…Covalent labeling represents another important approach for monitoring protein conformations and interactions [14][15][16][17][18][19]. Solvent-accessible side chains can be modified by watersoluble reactive species, whereas buried segments are sterically protected.…”

Section: Introductionmentioning

confidence: 99%

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

Vahidi

Konermann

2016

J. Am. Soc. Mass Spectrom.

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Time (s)0Bleaching of the reporter dye cyanine-5 (Cy5) served as readout of the timedependent radical milieu. Surprisingly, Cy5 oxidation extends over tens of milliseconds. This time range is four orders of magnitude longer than expected from the FPOP literature. We demonstrate that the glutamine scavenger generates metastable secondary radicals in the FPOP solution, and that these radicals lengthen the time frame of Cy5 oxidation. Cy5 and similar dyes are widely used for monitoring the radical dose experienced by proteins in solution. The measured Cy5 kinetics thus strongly suggest that protein oxidation in FPOP extends over a much longer time window than previously thought (i.e., many milliseconds instead of one microsecond). The optical approach developed here should be suitable for assessing the performance of future FPOP-like techniques with improved temporal labeling characteristics.

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Combining Chemical Labeling, Bottom-Up and Top-Down Ion-Mobility Mass Spectrometry To Identify Metal-Binding Sites of Partially Metalated Metallothionein

Cited by 42 publications

References 69 publications

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

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