Dynamic light-scattering analysis of full-length human RPA14/32 dimer

Purification, crystallization and self-association

J. E. Habel, J. F. Ohren, Gloria E Borgstahl

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Replication protein A (RPA) is a single-stranded DNA-binding protein involved in all aspects of eukaryotic DNA metabolism. A soluble heterodimeric form of RPA is composed of 14 and 32 kDa subunits (RPA14/32). Dynamic light-scattering (DLS) analysis was used to improve the purification, stabilization and crystallization of RPA14/32. Increasing the concentration of reducing agent in the last stage of purification diminished the size of a secondary peak in the anion-exchange chromatograph and promoted a single species in solution. This resulted in decreased polydispersity in the purified protein and enhanced the crystallization time from 9-12 months to 6 d. With this homogeneous preparation, the reversible association of RPA14/32 into a dimer of dimers was demonstrated by DLS. Four different crystal forms of RPA14/32 were obtained for structure determination and complete diffraction data were collected using synchrotron radiation for three of them. Data to 2.4 Å resolution was collected from hexagonal crystals (P32 or P31; a = b = 63.0, c = 272.6Å) and to 2.2 and 1.9Å resolution from two orthorhombic crystal forms (both P212121; form I, a = 61.4, b = 75.2, c = 131.6 Å; form II, a = 81.8, b = 140.4, c = 173.1 Å).

Original languageEnglish (US)
Pages (from-to)254-259
Number of pages6
JournalActa Crystallographica Section D: Biological Crystallography
Volume57
Issue number2
DOIs
StatePublished - Feb 22 2001

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Replication Protein A
Dynamic light scattering
Crystallization
purification
Dimers
Purification
light scattering
dimers
Association reactions
crystallization
proteins
Crystals
Synchrotrons
Reducing Agents
DNA-Binding Proteins
Anions
deoxyribonucleic acid
Polydispersity
Radiation
crystals

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biophysics
  • Condensed Matter Physics
  • Structural Biology

Cite this

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title = "Dynamic light-scattering analysis of full-length human RPA14/32 dimer: Purification, crystallization and self-association",
abstract = "Replication protein A (RPA) is a single-stranded DNA-binding protein involved in all aspects of eukaryotic DNA metabolism. A soluble heterodimeric form of RPA is composed of 14 and 32 kDa subunits (RPA14/32). Dynamic light-scattering (DLS) analysis was used to improve the purification, stabilization and crystallization of RPA14/32. Increasing the concentration of reducing agent in the last stage of purification diminished the size of a secondary peak in the anion-exchange chromatograph and promoted a single species in solution. This resulted in decreased polydispersity in the purified protein and enhanced the crystallization time from 9-12 months to 6 d. With this homogeneous preparation, the reversible association of RPA14/32 into a dimer of dimers was demonstrated by DLS. Four different crystal forms of RPA14/32 were obtained for structure determination and complete diffraction data were collected using synchrotron radiation for three of them. Data to 2.4 {\AA} resolution was collected from hexagonal crystals (P32 or P31; a = b = 63.0, c = 272.6{\AA}) and to 2.2 and 1.9{\AA} resolution from two orthorhombic crystal forms (both P212121; form I, a = 61.4, b = 75.2, c = 131.6 {\AA}; form II, a = 81.8, b = 140.4, c = 173.1 {\AA}).",
author = "Habel, {J. E.} and Ohren, {J. F.} and Borgstahl, {Gloria E}",
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T1 - Dynamic light-scattering analysis of full-length human RPA14/32 dimer

T2 - Purification, crystallization and self-association

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AU - Ohren, J. F.

AU - Borgstahl, Gloria E

PY - 2001/2/22

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N2 - Replication protein A (RPA) is a single-stranded DNA-binding protein involved in all aspects of eukaryotic DNA metabolism. A soluble heterodimeric form of RPA is composed of 14 and 32 kDa subunits (RPA14/32). Dynamic light-scattering (DLS) analysis was used to improve the purification, stabilization and crystallization of RPA14/32. Increasing the concentration of reducing agent in the last stage of purification diminished the size of a secondary peak in the anion-exchange chromatograph and promoted a single species in solution. This resulted in decreased polydispersity in the purified protein and enhanced the crystallization time from 9-12 months to 6 d. With this homogeneous preparation, the reversible association of RPA14/32 into a dimer of dimers was demonstrated by DLS. Four different crystal forms of RPA14/32 were obtained for structure determination and complete diffraction data were collected using synchrotron radiation for three of them. Data to 2.4 Å resolution was collected from hexagonal crystals (P32 or P31; a = b = 63.0, c = 272.6Å) and to 2.2 and 1.9Å resolution from two orthorhombic crystal forms (both P212121; form I, a = 61.4, b = 75.2, c = 131.6 Å; form II, a = 81.8, b = 140.4, c = 173.1 Å).

AB - Replication protein A (RPA) is a single-stranded DNA-binding protein involved in all aspects of eukaryotic DNA metabolism. A soluble heterodimeric form of RPA is composed of 14 and 32 kDa subunits (RPA14/32). Dynamic light-scattering (DLS) analysis was used to improve the purification, stabilization and crystallization of RPA14/32. Increasing the concentration of reducing agent in the last stage of purification diminished the size of a secondary peak in the anion-exchange chromatograph and promoted a single species in solution. This resulted in decreased polydispersity in the purified protein and enhanced the crystallization time from 9-12 months to 6 d. With this homogeneous preparation, the reversible association of RPA14/32 into a dimer of dimers was demonstrated by DLS. Four different crystal forms of RPA14/32 were obtained for structure determination and complete diffraction data were collected using synchrotron radiation for three of them. Data to 2.4 Å resolution was collected from hexagonal crystals (P32 or P31; a = b = 63.0, c = 272.6Å) and to 2.2 and 1.9Å resolution from two orthorhombic crystal forms (both P212121; form I, a = 61.4, b = 75.2, c = 131.6 Å; form II, a = 81.8, b = 140.4, c = 173.1 Å).

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