On the location of histones H1 and H5 in the chromatin fiber. Studies with immobilized trypsin and chymotrypsin.
The location of linker histones H1 and H5 in chicken erythrocyte chromatin was studied as a function of the fiber structure by the use of proteolytic enzymes immobilized onto Immobilon membranes. The immobilization of trypsin and chymotrypsin creates proteolytic probes, specific respectively to the terminal portions of the molecules or to the phenylalanine in the globular domain, that are incapable of penetrating into the interior of the condensed fiber. The chromatin fiber was studied in three different conformations: open zig-zag (in Tris buffer), closed zig-zag (upon addition of 10 mM-NaCl), or 30 nm fiber (upon addition of 0.35 mM-MgCl2). The results from digestion experiments performed on linker histones either in chicken erythrocyte chromatin, or free in solution or bound in mononucleosomes revealed several features relevant to linker histone location: (1) histone H5 is more protected than histone H1 in the fiber; (2) the N and C-terminal portions of histone H1 do not change their accessibility, and hence their location, upon compaction of the fiber; this behavior of H1 is in contrast to that of histone H5, whose tails become significantly internalized in the 30 nm fiber; (3) phenylalanine in the globular domain of both H1 and H5 is inaccessible (buried) both in the fiber and in the mononucleosomal particle. Sedimentation velocity measurements performed during the course of trypsin digestion demonstrate that the conformation of the fiber is highly sensitive to even a few cuts in some of the linker histone molecules; hence, the linker histones are an important factor in the organization of the fiber in all its different condensation states.
Leuba
S H
SH
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305.
Zlatanova
J
J
van Holde
K
K
D000818:Animals; D002498:Centrifugation; D002645:Chickens; D002843:Chromatin / Q000737:chemistry*; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D006657:Histones / Q000032:analysis*; D007700:Kinetics; D011487:Protein Conformation; D012996:Solutions; D014357:Trypsin
D002843:Chromatin; D004800:Enzymes, Immobilized; D006657:Histones; D012996:Solutions; D002918:Chymotrypsin; D014357:Trypsin