Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function

Shirley Y. Lee, Lester Pullen, Daniel J. Virgil, Carlos A. Castañeda, Dulith Abeykoon, Daniel N.A. Bolon, David Fushman

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.

Original languageEnglish (US)
Pages (from-to)1377-1389
Number of pages13
JournalJournal of Molecular Biology
Issue number7
StatePublished - Apr 3 2014
Externally publishedYes


  • proteasome
  • protein function
  • structural dynamics
  • thermodynamic stability
  • ubiquitin recycling

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


Dive into the research topics of 'Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function'. Together they form a unique fingerprint.

Cite this