The action of chymotrypsin on nucleosome cores. Histone products and conformational effects of limited digestion.
alpha-Chymotrypsin was used to probe accessible hydrophobic amino acid residues in nucleosome cores. Small amounts of chymotrypsin rapidly and selectively cleaved at leucine 20 of histone H3. Cleavage at this site caused partial unfolding of the nucleosome core at low ionic strengths indicated by a small decrease in sedimentation coefficient and increase in circular dichroism in the 265-285-nm range. Unfolding did not occur at moderate ionic strengths, probably because of more effective electrolyte screening of residual negative charge on the nucleosome core. More extensive treatment with chymotrypsin partially degraded other core histones in nucleosome cores at similar rates. The primary sites of cleavage were assigned to Leu115 of H2a, Val18 or Gln22 of H2b, and Leu10 plus Leu22 of H4. We conclude that these primary sites of chymotrypsin cleavage of the four core histones lie on or near the nucleosome core surface, while the large number of other hydrophobic histone residues located in more central sequences must be inaccessible. Extensive chymotrypsin treatment yielded a set of "limit" products approximately 80-100 residues long that were similar to the limit products of trypsin digestion. Sedimentation coefficients and circular dichroism spectra of nucleosome cores treated to near limits with chymotrypsin or chymotrypsin followed by trypsin were not consistent with significant unfolding of the proteolyzed cores at moderate ionic strength. These results indicate that the amino-terminal 20-30 residues of H2b, H3, and H4 and the amino- and carboxyl-terminal approximately 12 residues of H2a, in toto, interact weakly if at all with DNA of isolated nucleosome cores. These histone termini stabilize less than two turns and perhaps only one turn on each DNA terminus.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000032:analysis*; D009707:Nucleosomes / Q000378:metabolism*; D009994:Osmolar Concentration; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D006657:Histones; D009707:Nucleosomes; D002918:Chymotrypsin; D014357:Trypsin