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Structural and functional characterization of the core single‐stranded DNA‐binding domain of purine‐rich element binding protein B (Purβ)
Author(s) -
Rumora Amy E.,
Ramsey Jon E.,
Ballif Bryan A.,
Kelm Robert J.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.678.18
Subject(s) - circular dichroism , dna , recombinant dna , chemistry , binding domain , dna binding domain , enhancer , protein secondary structure , biochemistry , binding site , microbiology and biotechnology , electrophoretic mobility shift assay , biophysics , biology , transcription factor , gene
Purβ is a sequence‐specific single‐stranded DNA (ssDNA)‐binding protein that represses smooth muscle α‐actin gene transcription in stress‐activated fibroblasts and smooth muscle cells via cooperative interaction with two sites in the purine‐rich strand of a 5′ enhancer element (dubbed PE32‐F). To better understand how the modular structure of Purβ dictates its specific ssDNA‐binding function, we evaluated the interaction of purified recombinant Purβ with PE32‐F using spectroscopic and biochemical approaches. Results of intrinsic protein fluorescence and circular dichroism analyses pointed to a ssDNA‐induced change in protein conformation. Consistent with this interpretation, Purβ was protected from proteolysis when complexed with PE32‐F. Limited tryptic digestion of Purβ liberated a core ~30 kDa fragment spanning residues 29‐305 as determined by mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to dimerize. Quantitative ssDNA‐binding assays revealed that the core domain binds to PE32‐F with similar specificity but increased affinity compared to full‐length Purβ and a truncated 37‐263 mutant absent a putative amphipathic helix‐forming module. These findings provide new insight into the structural basis for the specific and high affinity binding of Purβ to ssDNA. NIH R01 HL054281 , T32 HL007594 , P20 RR16462.