Solid-Phase Peptide Synthesis

Fields, Gregg B.

doi:10.1007/978-1-59259-642-3_42

Cited by 13 publications

(15 citation statements)

References 106 publications

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“…Calculations of absolute surface area (ASA) of each residue in apo-MT2 obtained by MD after 4 ns showed cysteine residues from Cys6 up to Cys20 and from Cys34 to Cys42 to be less accessible for solvent (Table S4, ESI †). These results are in agreement with those obtained from tryptic digestion and match fragments [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and [32][33][34][35][36][37][38][39][40][41][42][43] in which alkylation was not completed, possibly due to the lower solvent accessibility.…”

Section: Alkylation Of Metal-free Metallothionein-2supporting

confidence: 81%

“…32,33 The resinattached peptide was cleaved from the resin by 2 h incubation with a mixture of TFA/thioanisole/EDT/anisole (90/5/3/2, v/v/v/v), followed by precipitation in cold (À20 1C) Et 2 O. The crude peptide was purified by HPLC (Dionex Ultimate 3000 system) on a Phenomenex C18 column (Gemini-NX 5 mm, 110 Å) using 0.1% TFA with a 0-45% ACN gradient in 30 min and then lyophilized.…”

Section: Synthesis Of B-domain Of Mt2mentioning

confidence: 99%

“…The MALDI spectrum recorded for unmodified MT2 demonstrates one major signal of 6043.7 m/z (Table S1 and [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and [32][33][34][35][36][37][38][39][40][41][42][43], but incomplete modification was also observed in other peptides (Table 1, Tables S2 and S3, ESI †). The pK a values of cysteine thiols predicted by PROPKA method revealed an acidity shift of Cys residues, which ranges from 6.9 to 11.9.…”

Section: Alkylation Of Metal-free Metallothionein-2mentioning

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: Alkylation Of Metal-free Metallothionein-2supporting

confidence: 81%

Section: Synthesis Of B-domain Of Mt2mentioning

confidence: 99%

Section: Alkylation Of Metal-free Metallothionein-2mentioning

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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“…Peptides were synthesized using Fmoc chemistry by a standard solidphase method [27] as described previously [7]; l-isomers of amino acids were used in all syntheses. One-letter annotation is used for denoting amino acids throughout this paper [28].…”

Section: Peptide Synthesismentioning

confidence: 99%

K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase

et al. 2005

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Histones are modified post‐translationally, e.g. by methylation of lysine and arginine residues, and by phosphorylation of serine residues. These modifications regulate processes such as gene expression, DNA repair, and mitosis and meiosis. Recently, evidence has been provided that histones are also modified by covalent binding of the vitamin biotin. The aims of this study were to identify biotinylation sites in histone H3, and to investigate the crosstalk among histone biotinylation, methylation and phosphorylation. Synthetic peptides based on the sequence of human histone H3 were used as substrates for enzymatic biotinylation by biotinidase; biotin in peptides was probed using streptavidin peroxidase. These studies provided evidence that K4, K9 and K18 in histone H3 are good targets for biotinylation; K14 and K23 are relatively poor targets. Antibodies were generated to histone H3, biotinylated either at K4, K9 or K18. These antibodies localized to nuclei in human placental cells in immunocytochemistry and immunoblotting experiments, suggesting that lysines in histone H3 are biotinylated in vivo. Dimethylation of R2, R8 and R17 increased biotinylation of K4, K9 and K18, respectively, by biotinidase; phosphorylation of S10 abolished biotinylation of K9. These observations are consistent with crosstalk between biotinylation of histones and other known modifications of histones. We speculate that this crosstalk provides a link to known roles for biotin in gene expression and cell proliferation.

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“…Novel systems and phenomena (2) have been created and observed at interfaces formed by hard materials, such as insulators and semiconductors. Concurrent with development of inorganic structures, methods for artificial bulk synthesis of biological macromolecules have been developed, particularly peptide-based species (3). Given these accomplishments in producing inorganic and biological materials, it is a natural extension of materials development to inquire about the interfaces that may be formed between them.…”

mentioning

confidence: 99%

Differential adhesion of amino acids to inorganic surfaces

Willett¹,

Baldwin²,

West³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

172

200

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A fundamental, yet underexplored, materials system is the interface between biological molecules and inorganic surfaces. In an elemental approach to this problem, we have systematically examined the adhesion of amino acids to a series of inorganic surfaces including metals, insulators, and semiconductors. Significant differential adhesion is observed over the full complement of amino acids, determined largely by amino acid side-chain charge. Extensive mapping of the amino acid adhesion versus materials in multiple solutions is presented, with preliminary mechanisms derived from concentration and pH dependence. These results provide an empirical basis for building peptide to inorganic surface structures, and, using this adhesion data, we design inorganic nanostructures that are shown to selectively bind to prescribed primary peptide sequences.peptide adhesion ͉ semiconductors

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Solid-Phase Peptide Synthesis

Cited by 13 publications

References 106 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

K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase

Differential adhesion of amino acids to inorganic surfaces

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