Interaction between DNA and chromosomal proteins HMGB1 and H1 studied by IR/VCD spectroscopy

Abstract

Binary complexes of calf thymus DNA with calf thymus non-histone chromosomal protein HMGB1 and linker histone H1 were studied using FTIR/VCD spectroscopy. The spectroscopic data showed that the interaction of the protein HMGB1 and histone H1 with DNA resulted in formation of two different types of the macromolecular complexes. Histone H1 retained its native structure even at high concentrations and induced DNA condensation upon binding at the protein to DNA ratio r (w/w) in the complex r ⩾ 0.3. HMGB1 demonstrated the ability to form soluble complexes at considerably higher protein to DNA ratios. The obtained data suggest that the HMGB1 also participated in the protein–protein interactions via its C-terminal domain.

Introduction

HMGB proteins are the family of chromatin proteins that contain the structural HMGB motif (HMGB-domain [1]) specific for the High Mobility Group proteins 1 and 2. The abundant members of this family are well known for their unusual DNA-binding properties characteristic for the motif [2]. These proteins are able to recognize a variety of structural distortions in the DNA double helix [3], [4], [5]. HMGB proteins are also able to induce bends of up to 140° in DNA upon interaction [6]. Although the particular role of these proteins remains unclear, they were assigned mostly structural functions. Reflecting this ability they were also called architectural factors of chromatin [7]. In some cases they act together with other proteins, which function as a part of rather big DNA–protein complexes [8], [9]. Protein–protein interactions affect strongly DNA-binding properties of HMGB1 especially in complex DNA–protein systems [10], [11], [12], [13], [14].

Histone H1 is one of the best studied chromatin proteins [15], [16], [17], [18]. It binds to linker DNA at the entrance/exit of the nucleosome. This interaction takes place through the major groove of DNA and results in DNA bending around the protein molecule. Both HMGB1 and H1 bind linker DNA in chromatin and there were several attempts to study their mutual influence on DNA binding [8], [19], [20], [21]. The data suggest that these two proteins demonstrate a co-action helping each other to some extent rather than competing in binding. There were several attempts to study the complexes using different spectroscopic techniques, including FTIR/VCD [11], [13], [14], [21], [22]. However, even the structures of the binary complexes between DNA and the individual proteins H1 or HMGB1 are not clear yet, which significantly complicates the interpretation of the spectral data for the ternary complexes.

Infrared or vibrational circular dichroism spectroscopy (VCD) is a relatively new tool for investigating the structure of biological macromolecules (see [23], [24] and references therein). Like its counterpart in the ultraviolet region (electronic CD or ECD), VCD is very sensitive to structural changes in the macromolecules. VCD on the other hand is considerably more informative for structural analysis of large supra molecular complexes of DNA with chromosomal proteins [21]. The present work is the first attempt to study binary complexes of the calf thymus DNA with natural calf thymus proteins HMGB1 and H1 using FTIR/VCD spectroscopic approach.