Interaction between DNA and chromosomal proteins HMGB1 and H1 studied by IR/VCD spectroscopy
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.
Section snippets
Experimental section
Calf thymus DNA (Sigma) was used with further sonication as described elsewhere [25]. All non-organic salts were of spectroscopic grade (Alfa Aesar). All aqueous solutions were prepared using double distilled deionized water. Heavy water (Sigma, 99.9% D2O) was used for the IR and VCD experiments. To obtain the DNA–protein complexes, histone H1 (MW 21000) and non-histone chromosomal protein HMGB1 (MW 26500) were used. Both proteins were isolated from calf thymus as described earlier [26]. The
Conformation of DNA and individual proteins
Artificial complexes DNA–H1 and DNA–HMGB1 were studied using FTIR absorption spectroscopy and vibrational circular dichroism (VCD). The IR absorption and VCD spectra of D2O solutions of calf thymus DNA are shown in Fig. 1. The figure also shows the noise estimate for VCD spectrum of DNA. The position and the assignment of the corresponding bands are summarized in Table 1. A detailed description of the IR/VCD spectra of calf thymus DNA and their band assignment can be found elsewhere [21], [25],
Conclusions
The FTIR absorption and circular dichroism spectra (VCD) were recorded and analyzed for the complexes of calf thymus DNA and two chromosomal proteins: linker histone H1 and non-histone chromosomal protein HMGB1. The spectroscopic data show that the interaction of the protein HMGB1 and histone H1 with DNA result in formation of two different types of the macromolecular complexes. Histone H1 retains its native structure even at high concentrations and induces DNA condensation upon binding at the
Acknowledgements
The authors acknowledge financial support from the Russian Foundation for Basic Research (RFBR) (Grant 12-08-01134) and Federal program ‘Scientific and pedagogical labor force for an innovative Russia’ (The Ministry of Education and Science of the Russian Federation). We are also grateful to Dr. H. Wieser (University of Calgary) for his help and support of this study.
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