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Titolo:
Identification of intrinsic order and disorder in the DNA repair protein XPA
Autore:
Iakoucheva, LM; Kimzey, AL; Masselon, CD; Bruce, JE; Garner, EC; Brown, CJ; Dunker, AK; Smith, RD; Ackerman, EJ;
Indirizzi:
Pacific NW Natl Lab, Mol Biosci Dept, Richland, WA 99352 USA Pacific NW Natl Lab Richland WA USA 99352 ci Dept, Richland, WA 99352 USA Battelle Mem Inst, Pacific NW Natl Labs, Environm Mol Sci Lab, Richland, WA 99352 USA Battelle Mem Inst Richland WA USA 99352 l Sci Lab, Richland, WA 99352 USA Washington State Univ, Sch Mol Biosci, Pullman, WA 99164 USA Washington State Univ Pullman WA USA 99164 Biosci, Pullman, WA 99164 USA
Titolo Testata:
PROTEIN SCIENCE
fascicolo: 3, volume: 10, anno: 2001,
pagine: 560 - 571
SICI:
0961-8368(200103)10:3<560:IOIOAD>2.0.ZU;2-0
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEOTIDE EXCISION-REPAIR; LIMITED PROTEOLYTIC SITES; MASS-SPECTROMETRY; XENOPUS-LAEVIS; BINDING DOMAIN; CRYSTAL-STRUCTURES; GLOBULAR-PROTEINS; BETA; CONFORMATION; COMPLEX;
Keywords:
XPA; mass spectrometry; intrinsic disorder; partial proteolysis; unstructured proteins; DNA repair;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
50
Recensione:
Indirizzi per estratti:
Indirizzo: Ackerman, EJ Pacific NW Natl Lab, Mol Biosci Dept, POB 999, Richland, WA 99352 USA Pacific NW Natl Lab POB 999 Richland WA USA 99352 A 99352 USA
Citazione:
L.M. Iakoucheva et al., "Identification of intrinsic order and disorder in the DNA repair protein XPA", PROTEIN SCI, 10(3), 2001, pp. 560-571

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 thismolecule 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 ofthe 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 thatdisorder 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 wasstrong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

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Documento generato il 10/04/20 alle ore 14:04:59