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J. Biol. Chem., Vol. 282, Issue 3, 1552-1560, January 19, 2007

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Hydrodynamic Studies on the Quaternary Structure of Recombinant Mouse Pur^*

Jon E. Ramsey, Margaret A. Daugherty, and Robert J. Kelm, Jr.^¶1

From the Departments of Biochemistry and ^¶Medicine, Cardiovascular Research Institute, University of Vermont College of Medicine, Burlington, Vermont 05405 and Department of Chemistry and Biochemistry, Middlebury College, Middlebury, Vermont 05753

Pur 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 Pur, Pur 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 Pur and Pur 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 Pur devoid of nucleic acid. Recombinant Pur 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 Pur reversibly self-associates in the absence of nucleic acid. Both approaches independently showed that the hydrodynamic shape of the Pur homodimer is markedly asymmetric and non-spherical. Sedimentation velocity analyses indicated that dimeric Pur has a sedimentation coefficient of 3.96 Svedberg, a frictional coefficient ratio (f/f₀) 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 Pur self-associates in a reversible monomer-dimer equilibrium characterized by a K_d = 1.13 ± 0.27 µM.

Received for publication, October 3, 2006 , and in revised form, November 20, 2006.

^* The study was supported by NHLBI, National Institutes of Health Grant HL54281 (to R. J. K.) and American Heart Association Predoctoral Fellowship 0515620T (to J. E. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material.

¹ To whom correspondence should be addressed: Colchester Research Facility, 208 South Park Dr., Colchester, VT 05446. Tel.: 802-656-0329; Fax: 802-656-8969; E-mail: Robert.Kelm{at}uvm.edu.

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This article has been cited by other articles:

				A. M. Knapp, J. E. Ramsey, S.-X. Wang, A. R. Strauch, and R. J. Kelm Jr. Structure-Function Analysis of Mouse Pur II: CONFORMATION ALTERING MUTATIONS DISRUPT SINGLE-STRANDED DNA AND PROTEIN INTERACTIONS CRUCIAL TO SMOOTH MUSCLE {alpha}-ACTIN GENE REPRESSION J. Biol. Chem., December 7, 2007; 282(49): 35899 - 35909. [Abstract] [Full Text] [PDF]