A versatile strategy for the semisynthetic production of Ser65 phosphorylated ubiquitin and its biochemical and structural characterisation

Cong Han, Kuan-Chuan Pao, Agne Kazlauskaite, Miratul M. K. Muqit, Satpal Virdee (Lead / Corresponding author)

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. 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.

Original languageEnglish
Pages (from-to)1574-1579
Number of pages6
JournalChemBioChem
Volume16
Issue number11
DOIs
Publication statusPublished - 27 Jul 2015

Fingerprint

Ubiquitin
Phosphorylation
Disulfides
Crystals
Ubiquitin-Protein Ligases
Parkinson Disease
Phosphotransferases
Chemical activation
Molecules

Keywords

  • Enzyme catalysis
  • Ligases
  • Phosphorylation
  • Synthetic methods
  • Ubiquitin

Cite this

@article{d0acc4d73b6246c288ff02916aa10050,
title = "A versatile strategy for the semisynthetic production of Ser65 phosphorylated ubiquitin and its biochemical and structural characterisation",
abstract = "Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. 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.",
keywords = "Enzyme catalysis, Ligases, Phosphorylation, Synthetic methods, Ubiquitin",
author = "Cong Han and Kuan-Chuan Pao and Agne Kazlauskaite and Muqit, {Miratul M. K.} and Satpal Virdee",
year = "2015",
month = "7",
day = "27",
doi = "10.1002/cbic.201500185",
language = "English",
volume = "16",
pages = "1574--1579",
journal = "ChemBioChem",
issn = "1439-4227",
publisher = "Wiley-VCH",
number = "11",

}

A versatile strategy for the semisynthetic production of Ser65 phosphorylated ubiquitin and its biochemical and structural characterisation. / Han, Cong; Pao, Kuan-Chuan; Kazlauskaite, Agne; Muqit, Miratul M. K.; Virdee, Satpal (Lead / Corresponding author).

In: ChemBioChem, Vol. 16, No. 11, 27.07.2015, p. 1574-1579.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A versatile strategy for the semisynthetic production of Ser65 phosphorylated ubiquitin and its biochemical and structural characterisation

AU - Han, Cong

AU - Pao, Kuan-Chuan

AU - Kazlauskaite, Agne

AU - Muqit, Miratul M. K.

AU - Virdee, Satpal

PY - 2015/7/27

Y1 - 2015/7/27

N2 - Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. 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.

AB - Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. 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.

KW - Enzyme catalysis

KW - Ligases

KW - Phosphorylation

KW - Synthetic methods

KW - Ubiquitin

UR - http://www.scopus.com/inward/record.url?scp=84937253640&partnerID=8YFLogxK

U2 - 10.1002/cbic.201500185

DO - 10.1002/cbic.201500185

M3 - Article

C2 - 26010437

AN - SCOPUS:84937253640

VL - 16

SP - 1574

EP - 1579

JO - ChemBioChem

JF - ChemBioChem

SN - 1439-4227

IS - 11

ER -