Structural analysis of Trypanosoma brucei brucei chromatin by limited proteolysis. An electrical-birefringence study

The sensitive electric-birefringence method was used to reveal structural differences between the soluble chromatin of procyclic Trypanosoma brucei brucei and the chromatin of the higher eukaryotes. The orientation of the nucleosomal chains and the presence of extended DNA were analysed from the sign and amplitude of the steady-state birefringence, and the conformational properties (overall dimensions and flexibility) were studied in relation to the orientational relaxation times. In contrast to the higher eukaryotes, the birefringence of T. brucei brucei is negative and of low amplitude, corresponding to that of H1-depleted rat liver nucleosomes. Furthermore, the relaxation times are very small, about 10 microseconds. If salt is added, the birefringence as well as the relaxation time decreases dramatically, indicating that condensation affects T. brucei brucei chromatin although it behaves like nucleosome filaments, with less stable DNA-protein interaction than for the higher eukaryotes. However, this condensation does not induce the formation of regular higher-order structure. This complies with the hypothesis that typical histone H1 is absent from T. brucei brucei chromatin and that a protein or protein domain fulfils the role of histone H1. The accessibility and structural role of histone-like proteins in T. brucei brucei chromatin were also investigated using limited proteolysis with enzymes covalently bound to nylon spheres. The analysis of protein products obtained after digestion with immobilized trypsin and subtilisin shows that proteins a and d, which are classified as H3 and H4 histones, respectively, are the first to be attacked. The changes in chromatin conformation indicate that chromatin undergoes a structural transition, leading to decondensation, as indicated by increases in negative birefringence and relaxation time, and to a change in its orientation mechanism, indicated by the appearance of a permanent moment. This result is very interesting since, in rat liver, H4 was very resistant and was the last histone to be attacked, suggesting internal location and its involvement in nucleosome stabilization rather than higher-order condensation. Therefore, in T. brucei brucei chromatin, the characteristic properties of proteins a and d (their composition and interaction with DNA), as well as their external location on the nucleosome surface, suggest that if these proteins play a role similar to that played by H3 and H4 in higher eukaryotes, probably through their N-terminal regions and interaction either with DNA or protein domains, the mechanisms involved in chromatin compaction are quite different.(ABSTRACT TRUNCATED AT 400 WORDS)