Supermetallization of peptides and proteins during electrospray ionization

Kostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Indeykina, Maria I.; Kozin, Sergey A.; Makarov, Alexander А.; Nikolaev, E. N.

doi:10.1002/jms.3622

Cited by 31 publications

(27 citation statements)

References 57 publications

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“…This fact has been underlined recently by two recent reports proving that investigations of Zn(II)-peptide/ protein equilibria by ESI in the gas phase are not quantitative due to zinc deposition or protein supermetalation during analysis. 64,65 Nevertheless, such a dynamic picture shows that two possible Zn(II) binding pathways may occur ''clustered'' via cluster formation and ''beaded'' via formation of individual coordination sites prior to coalescence into a cluster, as was postulated in a recent study. 14 On the other hand, our results strongly support the model in which initial metal ion binding takes place only in the a-domain.…”

Section: Zn(ii) Binding To the A-domain Of Mt2 Is Sequential Under Cementioning

confidence: 99%

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) Binding To the A-domain Of Mt2 Is Sequential Under Cementioning

confidence: 99%

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

et al. 2018

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“…Our results of Zn 2+ binding to T performed in the presence of Zn 2+ donors with various affinities (unpublished data) demonstrate that metal binding analysis performed by ESI-MS does not assess completely the equilibria present in solutions. Two recent papers published in mass spectrometry journals prove that investigations of Zn 2+ -equilibria by electrospray ionization in the gas phase are not quantitative due to zinc deposition or protein supermetallation during ESI-MS analysis [104,105]. …”

Section: Zn2+ Affinity For Human Metallothioneinmentioning

confidence: 99%

The Functions of Metamorphic Metallothioneins in Zinc and Copper Metabolism

Krężel

Maret

2017

IJMS

235

143

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Recent discoveries in zinc biology provide a new platform for discussing the primary physiological functions of mammalian metallothioneins (MTs) and their exquisite zinc-dependent regulation. It is now understood that the control of cellular zinc homeostasis includes buffering of Zn2+ ions at picomolar concentrations, extensive subcellular re-distribution of Zn2+, the loading of exocytotic vesicles with zinc species, and the control of Zn2+ ion signalling. In parallel, characteristic features of human MTs became known: their graded affinities for Zn2+ and the redox activity of their thiolate coordination environments. Unlike the single species that structural models of mammalian MTs describe with a set of seven divalent or eight to twelve monovalent metal ions, MTs are metamorphic. In vivo, they exist as many species differing in redox state and load with different metal ions. The functions of mammalian MTs should no longer be considered elusive or enigmatic because it is now evident that the reactivity and coordination dynamics of MTs with Zn2+ and Cu+ match the biological requirements for controlling—binding and delivering—these cellular metal ions, thus completing a 60-year search for their functions. MT represents a unique biological principle for buffering the most competitive essential metal ions Zn2+ and Cu+. How this knowledge translates to the function of other families of MTs awaits further insights into the specifics of how their properties relate to zinc and copper metabolism in other organisms.

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“…Recently, several groups have been focused on the use of the in‐ionization source H/D exchange for the enumeration of the labile hydrogens in the complex organic mixtures, peptides, proteins, oligosaccharides, and oligonucleotides . It was shown that high temperature of desolvating capillary can lead to such exotic effects as deuterium exchange at non‐labile sites, gas‐phase 16 O/ 18 O exchange, and formation of complexes of proteins with unusually high number of metals . Previously, investigating the H/D exchange of the proteins in the standard Thermo LTQ interface, we have encountered the impossibility to achieve the good degree of the reaction even at the highest temperature the capillary could be heated to (450 °C).…”

Section: Introductionmentioning

confidence: 99%

Thermal dissociation of ions limits the degree of the gas‐phase H/D exchange at the atmospheric pressure

et al. 2017

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We present the application of the extended desolvating capillaries for increasing the degree of the gas-phase hydrogen/deuterium exchange reaction at atmospheric pressure. The use of the extended capillaries results in the increase of the time that ions spend in the high pressure region, what leads to the significant improvement of the efficiency of the reaction. For the small protein ubiquitin, it was observed that for the same temperature, the number of exchanges increases with the decrease of the charge state so that the lowest charge state can exchange twice the number of hydrogen than the highest one. With the increase of the temperature, the difference decreases, and eventually, the number of exchanges equalizes for all charge states. The value of this temperature and the corresponding number of exchanges depend on the geometric parameters of the capillary. Further increase of the temperature leads to the thermal dissociation of the protein ion. The observed b/y fragments are identical to those produced by collision-induced dissociation performed in the ion trap. Copyright © 2017 John Wiley & Sons, Ltd.

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Supermetallization of peptides and proteins during electrospray ionization

Cited by 31 publications

References 57 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

The Functions of Metamorphic Metallothioneins in Zinc and Copper Metabolism

Thermal dissociation of ions limits the degree of the gas‐phase H/D exchange at the atmospheric pressure

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