Determination of the pKa of the N-terminal amino group of ubiquitin by NMR
Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules...
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Veröffentlicht in: | Scientific reports 2017-03, Vol.7 (1), p.43748-43748, Article 43748 |
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Zusammenfassung: | Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules to either one of the seven lysine residues of ubiquitin, or via its N-terminal α-amino group to build linear ubiquitin chains. The pK
a
of lysine side chains is around 10.5 and hence E3 ligases require a mechanism to deprotonate the amino group at physiological pH to produce an effective nucleophile. In contrast, the pK
a
of N-terminal α-amino groups of proteins can vary significantly, with reported values between 6.8 and 9.1, raising the possibility that linear chain synthesis may not require a general base. In this study we use NMR spectroscopy to determine the pK
a
for the N-terminal α-amino group of methionine1 of ubiquitin for the first time. We show that it is 9.14, one of the highest pK
a
values ever reported for this amino group, providing a rational for the observed need for a general base in the E3 ligase HOIP, which synthesizes linear ubiquitin chains. |
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ISSN: | 2045-2322 2045-2322 |
DOI: | 10.1038/srep43748 |