Salt effects on histone subunit interactions as studied by fluorescence spectroscopy

Abstract

The salt concentration dependence of the aggregation properties of calf thymus and chicken erythrocyte histones has been investigated by using fluorescence spectroscopy. The isolated H2A/H2B and H3/H4 subunit preparations were labeled with 5-(dimethylamino)naphthalene-1- sulfonyl (dansyl). This long-lived fluorescence probe allows for the observation of rotations due to tumbling of the particle and thus is a probe for changes in the size of macromolecular assemblies. The fluorescence polarization and lifetime were measured as a function of salt concentration for these isolated preparations. Next, each labeled preparation was reconstituted with its unlabeled complement, and the salt concentration dependence of histone core octamer interactions was investigated in the same manner. Salt-induced core particle formation was observed by monitoring the dansyl-labeled dimers for both the calf thymus and chicken erythrocyte preparations. Evidence for subunit dissociation of the isolated H2A-H2B preparations was also found, as well as aggregation of the isolated H3/H4 subunits to at least dimers of tetramers. The calf thymus H3/H4 preparation was in aggregated form under all conditions studied, whereas the chicken erythrocyte H3/H4 only formed aggregates at high protein or salt concentrations. We have found evidence that the dimer can displace the tetramer from the higher order aggregate in order to form core particles. Such competition between the subunit interfaces in the histone system suggests that they may play a regulatory role in histone-DNA interactions.

Histone subunit interactions as investigated by high pressure

“…Lowering the salt concentration to 200 mM slightly stabilized the protein to 6 X 10~8 M. Our data indicate a small negative volume change for the dissociation of the core particle octamer. The (H3)2(H4)2 tetramer, as was shown in the previous paper (Royer et al, 1989), also formed predominantly dimers of tetramers at higher protein or salt concentrations. In the study presented here, we found the dissociation constant for the H3/H4 octamer to dimer transition to be 1 X 10~21 M3 ***(Cly-2 = 4 X 10~8 M) at 2 M NaCl for the CT preparation.…”

Lifetime of the histone octamer studied by continuous-flow quasielastic light scattering: Test of a model for nucleosome transcription

“…Moreover, the apparent Stokes diameter obtained from the fits increased with the time of equilibration over a time scale of tens of minutes, ultimately reaching values that were significantly in excess of that determined for the octamer itself. It is known that the H2A-H2B heterodimers and the H32H42 tetramers produced by dissociation of histone octamers can form unnatural aggregates in approximately physiological ionic strength (Baxevanis et al, 1991;Royer et al, 1989;Scarlata et al, 1989;Sperling & Bustin, 1975; van Holde, 1989). We suspected that some of the heterodimers or tetramers or both might be polymerizing over time and that the autocorrelation functions obtained included contributions from both aggregates and any remaining heterodimers and tetramers.…”

“…An important attribute of this model is that it allows for maintenance of a relationship between regulatory signals encoded in the form of posttranslational histone modifications (van Holde, 1989;Wassarman & Kornberg, 1989) and the underlying DNA sequence. However, histone octamers are unstable in physiological ionic conditions; at equilibrium, octamers dissociate into an H3jH42 tetramer and two H2A-H2B heterodimers (Thomas & Butler, 1977; van Holde, 1989;Wassarman & Kornberg, 1989), and these in turn can repolymerize into nonnative H3-H4 or H2A-H2B polymers (Baxevanis et al, 1991;Royer et al, 1989;Scarlata et al, 1989;Sperling & Bustin, 1975; van Holde, 1989). If histone octamers were to dissociate during the time scale of polymerase progression, they would not be able to re-form a nucleosome after the polymerase had passed by.…”

Implementation of an experimental and computational tool set to study protein‐mAb interactions

“…FP has also been used to study the dynamics of monomer/dimer formation . Royer et al have studied the effect of salt concentration on histone subunit interactions using fluorescence polarization …”

“…[25][26][27] Royer et al have studied the effect of salt concentration on histone subunit interactions using fluorescence polarization. 28,29 Self-interaction chromatography (SIC) is a technique that can provide information about the self-interactions of a given protein, the second virial coefficient (B 22 ) and the effect of mobile phase modifiers on these interactions. 30 Cross interaction chromatography (CIC) is a modified form of SIC which enables one to study weak interactions between two different proteins.…”