Fourier transform infrared/vibrational circular dichroism spectroscopy as an informative tool for the investigation of large supramolecular complexes of biological macromolecules

Abstract

A combination of ultraviolet (UV) and infrared (IR) absorption and circular dichroism (CD) spectroscopy was applied to investigate the structure and formation of large supramolecular DNA-protein complexes. This combination of techniques was used to overcome limitations of UV-CD (electronic, or ECD) spectroscopy due to considerable light scattering in such solutions. Based on the analysis of FTIR and UV-CD spectra, the interaction of DNA with nonhistone chromatin protein HMGB1 and linker histone H1 was studied. The data obtained showed that under the conditions of the experiment (15 mM NaCl, protein/DNA ratio r < 1 w/w) the proteins did not reveal any AT or GC specificity in binding to DNA. In the presence of both proteins, mainly interactions in the DNA minor groove were observed, which were attributed to HMGB1 binding. Histone H1 facilitated binding of HMGB1 to DNA by interacting with the negatively charged groups of the sugar-phosphate backbone and binding of aspartic and glutamic amino acid residues of HMGB1. Acting together, HMGB1 and H1 stimulated the assemblage of supramolecular DNA-protein structures. The structural organization of the ternary complexes depended not only on the properties of the protein-DNA interactions but also on the interactions between HMGB1 and H1 molecules.

The effect of manganese(II) on the structure of DNA/HMGB1/H1 complexes: Electronic and vibrational circular dichroism studies

“…The spectral behavior of the complexes in the UV region is different for the Mn 2þ -DNA-protein complex than for the DNA-protein 36,43,68,69 and Mn 2þ -DNA 52 complexes and depends on the protein to DNA ratio. The ECD spectra of the complexes indicate that several structural stages are occurring in the system, depending on the protein to DNA ratio (Figure 3), and supramolecular structures are formed during these stages.…”

“…However, it is possible to obtain additional information by IR/VCD, since it enables one to study particles several orders of magnitude larger without considerable influence of light scattering. 43,61,62,70 Without the proteins, the presence of Mn 2þ leads to a significant intensity increase of almost all IR bands in the phosphate region, 52 which reflects the strong interaction of the metal ions with the sugarphosphate backbone. Addition of the protein to DNA without the presence of Mn 2þ produces no changes in the IR spectrum of DNA in this region ( Figure 5).…”

“…43,52 The IR and VCD spectra of the DNA and DNA-HMGB1/H1 complex in the region 1750-1450 cm À1 are shown in Figure 4. The absorption spectrum of the DNA-protein complex at r ¼ 0.1 and R ¼ 0.0, while generally similar to that of DNA alone, shows an increased intensity of the main composite band, which also contains an absorption from the proteins in the amide I region 62,66 at *1645 cm À1 and contributes to the intensity of the 1648-cm À1 DNA band.…”

“…Moreover, as the wavelength of IR radiation is longer, compared to UV, one can also study large complexes without significant contributions of light scattering. 43 VCD is a relatively new tool for investigating the structure of biological macromolecules. [44][45][46] It is very sensitive to structural changes in the macromolecules and considerably more informative for structural analysis compared to ECD.…”

Discrimination of human stem cells by photothermal microspectroscopy

“…The main wavenumbers responsible for the separation of spectra from SCs and differentiated cell types are those in the region of 1710-1700 cm -1 (figures 6,7 and 8), around of 1590 cm -1 (figures 7 and 8), the region of 1480 cm -1 ( figure 7 and 8 is associated with sugar ring vibrations 26 and C-O and C-C stretch in DNA 24 .…”