Dynamic light-scattering analysis of full-length human RPA14/32 dimer: purification, crystallization and self-association.
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 A resolution was collected from hexagonal crystals (P3(2) or P3(1); a = b = 63.0, c = 272.6 A) and to 2.2 and 1.9 A resolution from two orthorhombic crystal forms (both P2(1)2(1)2(1); form I, a = 61.4, b = 75.2, c = 131.6 A; form II, a = 81.8, b = 140.4, c = 173.1 A).
Habel
J E
JE
University of Toledo, Department of Chemistry, 2801 West Bancroft Street, Toledo, OH 43606, USA.
Ohren
J F
JF
Borgstahl
G E
GE
D003460:Crystallization; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D006801:Humans; D008027:Light; D021122:Protein Subunits; D011506:Proteins / Q000737:chemistry*; D012318:RNA Polymerase I; D011994:Recombinant Proteins / Q000737:chemistry; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D012542:Scattering, Radiation
D004268:DNA-Binding Proteins; D021122:Protein Subunits; D011506:Proteins; C497167:RPA1 protein, human; C093847:RPA14 protein, S cerevisiae; D011994:Recombinant Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D012318:RNA Polymerase I