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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

Expressed protein ligation and characterisation of phospho and non-phospho forms of Ub. A) Representative analytical HPLC of the ligation between Ub-1-45-SR (peak B) and UbC46-76 (peak A). The product, UbC46 (ubiquitin containing the Ala46Cys mutation), is observed after 5 min (peak C), and after 2 h the reaction has gone to near completion. B) HPLC analysis of UbC-pSer65 ligation product after purification by semi-preparative HPLC. C) ESI-MS spectrum of purified UbC46-pSer65; inset: deconvoluted spectrum (calcd: 8676.9 Da; found 8675 Da). D) ESI-MS spectrum for purified UbC46; inset, deconvoluted spectrum (calcd: 8596.9 Da; found 8595 Da). E) HPLC analysis of native Ub-pSer65 ligation product after free radical desulfurisation of UbC46-pSer65 for 3 h. F) ESI-MS spectrum of native Ub-pSer65 generated by desulfurisation. A single product (loss of 32 Da relative to UbC46-pSer65, 8675 Da) indicates quantitative conversion and preservation of the phosphate moiety (calcd: 8644.8 Da; found: 8643 Da).
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fig01: Expressed protein ligation and characterisation of phospho and non-phospho forms of Ub. A) Representative analytical HPLC of the ligation between Ub-1-45-SR (peak B) and UbC46-76 (peak A). The product, UbC46 (ubiquitin containing the Ala46Cys mutation), is observed after 5 min (peak C), and after 2 h the reaction has gone to near completion. B) HPLC analysis of UbC-pSer65 ligation product after purification by semi-preparative HPLC. C) ESI-MS spectrum of purified UbC46-pSer65; inset: deconvoluted spectrum (calcd: 8676.9 Da; found 8675 Da). D) ESI-MS spectrum for purified UbC46; inset, deconvoluted spectrum (calcd: 8596.9 Da; found 8595 Da). E) HPLC analysis of native Ub-pSer65 ligation product after free radical desulfurisation of UbC46-pSer65 for 3 h. F) ESI-MS spectrum of native Ub-pSer65 generated by desulfurisation. A single product (loss of 32 Da relative to UbC46-pSer65, 8675 Da) indicates quantitative conversion and preservation of the phosphate moiety (calcd: 8644.8 Da; found: 8643 Da).

Mentions: In parallel, Ub-1-45-SR was ligated to UbC46-76 and UbC46-76-Ser65 in denaturing phosphate buffer by using mercaptophenylacetic acid (MPAA) as catalyst.19 Ligation went to near completion after 2 h incubation at 25 °C (as determined by LC-MS) to yield ubiquitin with an Ala46Cys mutation (UbC46), or Ala46Cys and phosphoserine at position 65 (UbC46-pSer65; Figure 1 A). The products were purified by reversed phase HPLC and characterised by LC-MS (Figure 1 B–D). Lyophilised UbC46 and UbC46-pSer65 were obtained in 44 % yield. The polypeptides were then dissolved in denaturing buffer and folded by dialysis against non-denaturing buffer (yield ∼95 %). In-gel tryptic digestion and LC-MS/MS analysis confirmed phosphoserine at Ser65 (Figure S4). The yield of phosphoubiquitin obtained by enzymatic phosphorylation10 was not reported; however, a chromatographic step was required to remove kinase and unphosphorylated material. As our ligation went to near completion and a single chromatographic step was required, the yield from our approach most likely compares favourably.


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)

Expressed protein ligation and characterisation of phospho and non-phospho forms of Ub. A) Representative analytical HPLC of the ligation between Ub-1-45-SR (peak B) and UbC46-76 (peak A). The product, UbC46 (ubiquitin containing the Ala46Cys mutation), is observed after 5 min (peak C), and after 2 h the reaction has gone to near completion. B) HPLC analysis of UbC-pSer65 ligation product after purification by semi-preparative HPLC. C) ESI-MS spectrum of purified UbC46-pSer65; inset: deconvoluted spectrum (calcd: 8676.9 Da; found 8675 Da). D) ESI-MS spectrum for purified UbC46; inset, deconvoluted spectrum (calcd: 8596.9 Da; found 8595 Da). E) HPLC analysis of native Ub-pSer65 ligation product after free radical desulfurisation of UbC46-pSer65 for 3 h. F) ESI-MS spectrum of native Ub-pSer65 generated by desulfurisation. A single product (loss of 32 Da relative to UbC46-pSer65, 8675 Da) indicates quantitative conversion and preservation of the phosphate moiety (calcd: 8644.8 Da; found: 8643 Da).
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Related In: Results  -  Collection

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fig01: Expressed protein ligation and characterisation of phospho and non-phospho forms of Ub. A) Representative analytical HPLC of the ligation between Ub-1-45-SR (peak B) and UbC46-76 (peak A). The product, UbC46 (ubiquitin containing the Ala46Cys mutation), is observed after 5 min (peak C), and after 2 h the reaction has gone to near completion. B) HPLC analysis of UbC-pSer65 ligation product after purification by semi-preparative HPLC. C) ESI-MS spectrum of purified UbC46-pSer65; inset: deconvoluted spectrum (calcd: 8676.9 Da; found 8675 Da). D) ESI-MS spectrum for purified UbC46; inset, deconvoluted spectrum (calcd: 8596.9 Da; found 8595 Da). E) HPLC analysis of native Ub-pSer65 ligation product after free radical desulfurisation of UbC46-pSer65 for 3 h. F) ESI-MS spectrum of native Ub-pSer65 generated by desulfurisation. A single product (loss of 32 Da relative to UbC46-pSer65, 8675 Da) indicates quantitative conversion and preservation of the phosphate moiety (calcd: 8644.8 Da; found: 8643 Da).
Mentions: In parallel, Ub-1-45-SR was ligated to UbC46-76 and UbC46-76-Ser65 in denaturing phosphate buffer by using mercaptophenylacetic acid (MPAA) as catalyst.19 Ligation went to near completion after 2 h incubation at 25 °C (as determined by LC-MS) to yield ubiquitin with an Ala46Cys mutation (UbC46), or Ala46Cys and phosphoserine at position 65 (UbC46-pSer65; Figure 1 A). The products were purified by reversed phase HPLC and characterised by LC-MS (Figure 1 B–D). Lyophilised UbC46 and UbC46-pSer65 were obtained in 44 % yield. The polypeptides were then dissolved in denaturing buffer and folded by dialysis against non-denaturing buffer (yield ∼95 %). In-gel tryptic digestion and LC-MS/MS analysis confirmed phosphoserine at Ser65 (Figure S4). The yield of phosphoubiquitin obtained by enzymatic phosphorylation10 was not reported; however, a chromatographic step was required to remove kinase and unphosphorylated material. As our ligation went to near completion and a single chromatographic step was required, the yield from our approach most likely compares favourably.

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