Identification of intrinsic order and disorder in the DNA repair protein XPA

Lilia M. Iakoucheva, Amy L. Kimzey, Christophe D. Masselon, James E. Bruce, Ethan C. Garner, Celeste J. Brown, A. Dunker, Richard D. Smith, Eric J. Ackerman

Research output: Contribution to journalArticle

82 Citations (Scopus)

Abstract

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

Original languageEnglish (US)
Pages (from-to)560-571
Number of pages12
JournalProtein Science
Volume10
Issue number3
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Proteolysis
Xenopus
DNA Repair
Repair
Trypsin
Mass spectrometry
DNA
Nuclear magnetic resonance
Intrinsically Disordered Proteins
Cyclotron resonance
Electrospray ionization
Proteins
Polyacrylamide Gel Electrophoresis
Mass Spectrometry
Fourier transforms
Cyclotrons
Nucleotides
Molecular weight
Ions
Neural networks

Keywords

  • DNA repair
  • Intrinsic disorder
  • Mass spectrometry
  • Partial proteolysis
  • Unstructured proteins
  • XPA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Iakoucheva, L. M., Kimzey, A. L., Masselon, C. D., Bruce, J. E., Garner, E. C., Brown, C. J., ... Ackerman, E. J. (2001). Identification of intrinsic order and disorder in the DNA repair protein XPA. Protein Science, 10(3), 560-571. https://doi.org/10.1110/ps.29401

Identification of intrinsic order and disorder in the DNA repair protein XPA. / Iakoucheva, Lilia M.; Kimzey, Amy L.; Masselon, Christophe D.; Bruce, James E.; Garner, Ethan C.; Brown, Celeste J.; Dunker, A.; Smith, Richard D.; Ackerman, Eric J.

In: Protein Science, Vol. 10, No. 3, 2001, p. 560-571.

Research output: Contribution to journalArticle

Iakoucheva, LM, Kimzey, AL, Masselon, CD, Bruce, JE, Garner, EC, Brown, CJ, Dunker, A, Smith, RD & Ackerman, EJ 2001, 'Identification of intrinsic order and disorder in the DNA repair protein XPA', Protein Science, vol. 10, no. 3, pp. 560-571. https://doi.org/10.1110/ps.29401
Iakoucheva LM, Kimzey AL, Masselon CD, Bruce JE, Garner EC, Brown CJ et al. Identification of intrinsic order and disorder in the DNA repair protein XPA. Protein Science. 2001;10(3):560-571. https://doi.org/10.1110/ps.29401
Iakoucheva, Lilia M. ; Kimzey, Amy L. ; Masselon, Christophe D. ; Bruce, James E. ; Garner, Ethan C. ; Brown, Celeste J. ; Dunker, A. ; Smith, Richard D. ; Ackerman, Eric J. / Identification of intrinsic order and disorder in the DNA repair protein XPA. In: Protein Science. 2001 ; Vol. 10, No. 3. pp. 560-571.
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