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A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation.

Han C, Pao KC, Kazlauskaite A, Muqit MM, Virdee S - Chembiochem (2015)

Bottom Line: Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form.The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function.Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).

No MeSH data available.


Related in: MedlinePlus

Strategy for expressed protein ligation (EPL) of site-specifically phosphorylated ubiquitin. The N-terminal fragment of Ub (residues 1–45) was obtained as a C-terminal thioester by thiolysis of a recombinant Ub1–45–intein fusion. The C-terminal peptide (residues 46–76 including phosphoserine at position 65) was synthesised commercially by solid-phase peptide synthesis. The native Ala46 residue was mutated to Cys to facilitate EPL; subsequent Cys desulfurisation furnishes the native protein.
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sch1: Strategy for expressed protein ligation (EPL) of site-specifically phosphorylated ubiquitin. The N-terminal fragment of Ub (residues 1–45) was obtained as a C-terminal thioester by thiolysis of a recombinant Ub1–45–intein fusion. The C-terminal peptide (residues 46–76 including phosphoserine at position 65) was synthesised commercially by solid-phase peptide synthesis. The native Ala46 residue was mutated to Cys to facilitate EPL; subsequent Cys desulfurisation furnishes the native protein.


A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation.

Han C, Pao KC, Kazlauskaite A, Muqit MM, Virdee S - Chembiochem (2015)

Strategy for expressed protein ligation (EPL) of site-specifically phosphorylated ubiquitin. The N-terminal fragment of Ub (residues 1–45) was obtained as a C-terminal thioester by thiolysis of a recombinant Ub1–45–intein fusion. The C-terminal peptide (residues 46–76 including phosphoserine at position 65) was synthesised commercially by solid-phase peptide synthesis. The native Ala46 residue was mutated to Cys to facilitate EPL; subsequent Cys desulfurisation furnishes the native protein.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581463&req=5

sch1: Strategy for expressed protein ligation (EPL) of site-specifically phosphorylated ubiquitin. The N-terminal fragment of Ub (residues 1–45) was obtained as a C-terminal thioester by thiolysis of a recombinant Ub1–45–intein fusion. The C-terminal peptide (residues 46–76 including phosphoserine at position 65) was synthesised commercially by solid-phase peptide synthesis. The native Ala46 residue was mutated to Cys to facilitate EPL; subsequent Cys desulfurisation furnishes the native protein.
Bottom Line: Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form.The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function.Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).

No MeSH data available.


Related in: MedlinePlus