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Interaction of HMG proteins and H1 with hybrid PNA–DNA junctions
Author(s) -
Totsingan Filbert,
Bell Anthony J.
Publication year - 2013
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2342
Subject(s) - circular dichroism , dna , chemistry , nucleic acid , peptide , recombinant dna , dna binding protein , crystallography , biophysics , biochemistry , biology , gene , transcription factor
Abstract The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein‐binding surface of an immobilized four‐way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ‐PNA 1 and 4WJ‐PNA 3 ). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two‐state equilibrium between: (i) an open‐x state favored by absence of Mg 2+ , low salt, and protein binding, and (ii) a compact stacked‐x state favored by Mg 2+ . 4WJ‐PNA 3 , like J1, shifts readily from an open to stacked conformation in the presence of Mg +2 , while 4WJ‐PNA 1 does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ‐PNA 3 with nearly identical affinity ( K D s) and 4WJ‐PNA 1 with two‐fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.