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Comput Struct Biotechnol J. 2013 Sep 10;7:e201304006. doi: 10.5936/csbj.201304006. eCollection 2013.

Biochemical and biophysical characterization of recombinant yeast proteasome maturation factor ump1.

Author information

  • 1IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
  • 2Centre for Molecular and Structural Biomedicine, CBME/IBB, LA, Portugal ; Current address: Biophysics Section, Department of Life Sciences, Imperial College, London, UK.
  • 3IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal ; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal.
  • 4Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal ; Current address: BioFIG - Centre for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences, University of Lisboa, Campo Grande 1749-016 Lisboa, Portugal.
  • 5Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal.
  • 6Centre for Molecular and Structural Biomedicine, CBME/IBB, LA, Portugal ; Current address: Immunodiagnostic Systems, Core Technology, 10 Didcot Way, Boldon, NE35 9PD, UK.
  • 7Institute for Genetics, University of Cologne, Zülpicher Str. 47, D-50674 Cologne, Germany.
  • 8Centre for Molecular and Structural Biomedicine, CBME/IBB, LA, Portugal ; Institute for Genetics, University of Cologne, Zülpicher Str. 47, D-50674 Cologne, Germany.

Abstract

Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump is a short-lived maturation factor required for the efficient biogenesis of the 20S proteasome. Upon the association of the two precursor complexes, Ump is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. In order to further understand the mechanisms behind proteasomal maturation, we expressed and purified yeast Ump in E. coli for biophysical and structural analysis. We show that recombinant Ump is purified as a mixture of different oligomeric species and that oligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein. Furthermore, a combination of bioinformatic, biochemical and structural analysis revealed that Ump shows characteristics of an intrinsically disordered protein, which might become structured only upon interaction with the proteasome subunits.

KEYWORDS:

Circular dichroism; dynamic light scattering; intrinsically disordered; protein structure

PMID:
24688736
[PubMed]
PMCID:
PMC3962104
Free PMC Article
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