A. M. Polyanichko

The HMG1 ta(i)le.

Abstract ‘You promised to tell me your history, you know,’ said Alice, ‘and why it is you hate—C and D,’ she added in a whisper, half afraid that it would be offended again. ‘Mine is a long and a sad tale!’ said the Mouse, turning to Alice, and sighing. ‘It IS a long tail, certainly,’ said Alice, looking down with wonder at the Mouses tail; ‘but why do you call it sad?’ And she kept on puzzling about it while the Mouse was speaking, so that her idea of the tale was something like this… “Alices Adventures in Wonderland” by Lewis Carroll We have studied structural changes in DNA/protein complexes using the CD spectroscopy, upon the interaction of HMG1-domains with calf thymus DNA at different ionic strengths. HMG1 protein isolated from calf thymus and recombinant HMG1-(A+B) protein were used. Recombinant protein HMG1-(A+B) represents a rat HMG1 lacking C-terminal acidic tail. At low ionic strength (15 mM NaCl) we observed similar behavior of both proteins upon interaction with DNA. Despite this, at higher ionic strength (150 mM NaCl) their interaction with DNA leads to a completely different structure of the complexes. In the case of HMG1-(A+B)/DNA complexes we observed the appearance of DNA fractions possessing very high optical activity. This could be a result of formation of the highly-ordered DNA structures modulated by the interaction with HMG1-domains. Thus the comparison studies of HMG1 and HMG1-(A+B) interaction with DNA show that negatively charged C-terminal tail of HMG1 modulates interaction of the protein with DNA. The striking difference of the behaviour of these two systems allows us to explain the functional role of multiple HMG1 domains in some regulatory and architectural proteins.

HMG1 Domains: The Victims of the Circumstances

The method of circular dichroism (CD) was used to compare DNA behavior during its interaction with linker histone H1 and with nonhistone chromosomal protein HMG1 at different ionic strength and at different protein content in the system. The role of the negatively charged C-terminal segment of HMG1 was analyzed using recombinant protein HMG1-(A+B), which lacks the C-terminal amino acid sequence. The ψ-type CD spectra were common for DNA interaction with histone H1, but no spectra of this type were observed in HMG1–DNA systems even at high ionic strength. The CD spectrum of the truncated recombinant protein at high salt concentration somewhat resembled the ψ+-type spectrum. Two very intense positive bands were located near 215 nm and near 272 nm, and the whole CD spectrum was positive. The role of the C-terminal part of HMG1 in the formation of ordered DNA–protein complexes is discussed.

Interaction of DNA with sperm-specific histones of the H1 family

Interactions of DNA with sperm-specific histones of the H1 family of sea urchin Strongylocentrotus intermedius, sea star Aphelasterias japonica, and bivalve mollusc Chlamis islandicus were studied using circular dichroism and the DNA melting analysis. Under physiological conditions, the highest DNA compacting ability was found in the echinoderm sperm H1 protein, in which additional α-helical domains are present in their C-terminal sequence. The derivative melting curves have two peaks: the low-temperature peak corresponds to the melting of free DNA, whereas the DNA regions bound to the protein melt at higher temperature. The highest stabilizing ability is characteristic of complexes with the mollusc sperm H1 protein.

C-terminal domain of nonhistone protein HMGB1 as a modulator of HMGB1–DNA structural interactions

The HMGB1 protein (High Mobility Group protein 1) participates in the formation of functionally significant DNA-protein complexes. HMGB1 protein contains two DNA-binding domains and negatively charged C-terminal region. The latter consists of continuous sequence of dicarboxylic amino acids residues. Structural changes in DNA-protein complexes were studied by circular dichroism spectroscopy (CD) and atomic force microscopy (AFM). Natural HMGB1 and recombinant protein HMGB1(A

Structural organization of DNA–protein complexes of chromatin studied by vibrational and electronic circular dichroism

Structure and functioning of chromatin is determined by interactions of DNA with numerous nuclear proteins. The most abundant and yet not completely understood non-histone chromosomal proteins are those belonging to a High Mobility Group (HMG) namely HMGB1. The interplay of this protein on DNA with linker histone H1 and other proteins determines both structure and functioning of the chromatin. A combination of UV and 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 (ECD) spectroscopy due to considerable light scattering in such solutions. Based on the analysis of FTIR and UV circular dichroism spectra and AFM imaging the interaction of DNA with high-mobility group non-histone chromatin protein HMGB1 and linker histone H1 was studied.

Analysis of the secondary structure of linker histone H1 based on IR absorption spectra

The effectiveness is compared of the infrared spectroscopy in the amide I region and UV circular dichroism to the analysis of the protein secondary structure by the example of the linker histone H1 and bovine serum albumin (BSA). It has been shown that the application of a diamond ATR cell gives the quantitative estimate of the fraction of α-helices and β-structures which are in a good agreement with UV circular dichroism spectroscopy. It has been shown that the histone H1 is able to aggregate, which results in considerable changes in its secondary structure.

Interaction between Nonhistone Protein HMGB1 and Linker Histone H1 Facilitates the Formation of Structurally Ordered DNA-Protein Complexes

The structural organization of the DNA complexes with nonhistone chromosomal protein and linker histone H1 was studied using circular dichroism spectroscopy (CD) and atomic force microscopy (AFM). It has been shown that due to the interaction between HMGB1 and H1 highly ordered DNA-protein complexes emerge in the solution. Their spectral properties are found to be similar to those of DNA/HMGB1-(AB) complexes, reported earlier. AFM images reveal the formation of fibril-like structures in the solution. We suggest that the electrostatic screening of the HMGB1 C-terminal domain by histone H1 facilitates stronger interaction of the HMGB1/H1 with DNA and the formation of the ordered supramolecular DNA-protein complexes.

Spectroscopic Study of the Interaction of DNA with the Linker Histone H1 from Starfish Sperm Reveals Mechanisms of the Formation of Supercondensed Sperm Chromatin

The interaction of the linker histone H1Z from the sperm chromatin of starfish Asterias amurensis with DNA was studied by spectroscopic and thermodynamic approaches. It has been shown that at the physiological conditions the interaction of the H1Z with DNA results in more compact structures compared to complexes of DNA with somatic histone H1. The typical profile of the DNA melting curves reveals two peaks attributed to the bound and unbound DNA. It has been shown that H1Z from starfish sperm stabilizes DNA to a greater extent compared to the somatic H1. It is possible that the presence of the additional α—helical segments within the C-terminal part of the H1Z typical for the linker histones from echinoderm sperm facilitates the protein-protein interactions which in turn stimulate cooperative binding of the histones to DNA, resulting in the formation of the supercompact sperm chromatin.

Structure of DNA complexes with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions: 1. Circular dichroism spectroscopy

Mechanisms of interaction of DNA with nonhistone chromosomal protein HMGB1 and linker histone H1 have been studied by means of circular dichroism and absorption spectroscopy. Both proteins are located in the internucleosomal regions of chromatin. It is demonstrated that the properties of DNA-protein complexes depend on the protein content and cannot be considered as a mere summing up of the effects of individual protein components. Interaction of the HMGB1 and H1 proteins is shown with DNA to be cooperative rather than competitive. Lysine-rich histone H1 facilitates the binding of HMGB1 to DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino acid residues in the C-terminal domain of HMGB1. The observed joint action of HMGB1 and H1 stimulates DNA condensation with the formation of anisotropic DNA-protein complexes with typical ψ-type CD spectra. Structural organization of the complexes depends not only on DNA-protein interactions but also on interaction between the HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the mode of interactions between components in the triple DNA-HMGB1-H1 complex. The binding of Mn2+ ions weakens DNA-protein interactions and strengthens protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.

Conformational properties of nuclear protein HMGB1 and specificity of its interaction with DNA

Changes in secondary structure of DNA and non-histone chromosomal protein HMGB1 during the formation of the complex have been studied by circular dichroism and UV spectroscopy. It was demonstrated that the HMGB1 protein is able to change its secondary structure upon binding to DNA. Based on the assumption that there are two spectroscopically distinguishable forms of the HMGB1 in solution, we estimated the fraction of bound protein. The fraction of bound protein decreases at higher protein to DNA ratios r from 0.48 at r = 0.13 to 0.06 at r = 2.43. It was shown that HMGB1 is able to induce considerable changes in DNA structure, even when the amount of protein actually bound is low.