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J Biol Chem. 2007 Jan 19;282(3):1552-60. Epub 2006 Nov 22.

Hydrodynamic studies on the quaternary structure of recombinant mouse Purbeta.

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  • 1Department of Biochemistry and Medicine, Cardiovascular Research Institute, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.

Abstract

Purbeta is a gene regulatory factor belonging to a family of highly conserved nucleic acid-binding proteins related by their ability to preferentially bind single-stranded DNA or RNA sequences rich in purine nucleotides. In conjunction with Puralpha, Purbeta has been implicated in transcriptional and translational repression of genes encoding contractile proteins found in the heart and vasculature. Although several models of sequence-specific DNA recognition, strand separation, and activator inhibition by oligomeric Puralpha and Purbeta have been proposed, it is currently unclear whether protein-protein interaction is a prerequisite to, or a consequence of nucleic acid binding. In this study, a recombinant protein purification scheme was devised to yield homogenous mouse Purbeta devoid of nucleic acid. Recombinant Purbeta was then subjected to light scattering and analytical ultracentrifugation analyses to assess the size, shape, and oligomeric state of the purified protein in solution. Results of laser light scattering and sedimentation velocity experiments indicated that Purbeta reversibly self-associates in the absence of nucleic acid. Both approaches independently showed that the hydrodynamic shape of the Purbeta homodimer is markedly asymmetric and non-spherical. Sedimentation velocity analyses indicated that dimeric Purbeta has a sedimentation coefficient of 3.96 Svedberg, a frictional coefficient ratio (f/f(0)) of 1.60, and a hydrodynamic radius of 4.43 nm. These values were consistent with those determined by independent dynamic light scattering studies. Sedimentation equilibrium analyses confirmed that Purbeta self-associates in a reversible monomer-dimer equilibrium characterized by a K(d) = 1.13 +/- 0.27 microm.

PMID:
17121857
[PubMed - indexed for MEDLINE]
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