Isolation and characterization of the core single-stranded DNA-binding domain of purine-rich element binding protein B (Purβ).
Purβ is a single-stranded nucleic acid-binding protein implicated in the injury-induced repression of genes encoding certain muscle-restricted isoforms of actin and myosin expressed in the heart, skeletal muscle, and vasculature. To better understand how the modular arrangement of the primary sequence of Purβ affects the higher order structure and function of the protein, purified recombinant Purβ was subjected to partial proteolysis in an attempt to identify a well-folded truncation protein that retained purine-rich single-stranded DNA-binding activity. Limited tryptic digestion of Purβ liberated a core ∼30kDa fragment corresponding to residues 29-305 as determined by epitope mapping and mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to self-associate while circular dichroism analysis confirmed that the Purβ core domain is stably folded in the absence of glycine-rich N- and C-terminal sequences. Comparative DNA-binding assays revealed that the isolated core domain interacts with purine-rich cis-elements from the smooth muscle α-actin gene with similar specificity but increased affinity compared to full-length Purβ. These findings suggest that the highly conserved modular repeats of Purβ fold to form a core functional domain, which mediates the specific and high affinity binding of the protein to single-stranded DNA.
Rumora
Amy E
AE
Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, USA.
Steere
Ashley N
AN
Ramsey
Jon E
JE
Knapp
Anna M
AM
Ballif
Bryan A
BA
Kelm
Robert J
RJ
D000818:Animals; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D051379:Mice; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014357:Trypsin / Q000737:chemistry
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C108770:Purb protein, mouse; D011994:Recombinant Proteins; D014357:Trypsin