Histone subunit interactions as investigated by high pressure.
High-pressure fluorescence polarization was used to investigate subunit interactions of the histone H2A-H2B dimer and the H3/H4 tetramer isolated from calf thymus (CT) and chicken erythrocyte (CE) chromatin. The proteins were individually labeled with the fluorescent probe 5-(dimethylamino)-naphthalene-1-sulfonate (dansyl or DNS), and the fluorescence polarization was measured as a function of pressure. The long fluorescence lifetime of the probe allows for the observation of global rotations of the protein, the rate of which is dependent upon the aggregation state. From the pressure dependence of the dansyl polarization, the Kd of H2A-H2B dissociation of the CE dimer was found to be approximately 1 X 10(-7) M at 2.0 M NaCl. 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 (C1/2 = 4 X 10(-8) M) at 2 M NaCl for the CT preparation. Decreasing the salt concentration to 200 mM reduced the stability of the CT H3/H4 octamer to 9 X 10(-21) M3 (C1/2 = 8 X 10(-8) M). The dimer of the CE tetramer also dissociated upon application of pressure in 2 M salt.(ABSTRACT TRUNCATED AT 250 WORDS)
Scarlata
S F
SF
College of Medicine, Cornell University Medical College, New York, New York 10021.
Ropp
T
T
Royer
C A
CA
D000818:Animals; D002417:Cattle; D002645:Chickens; D003619:Dansyl Compounds; D004912:Erythrocytes / Q000032:analysis; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006657:Histones / Q000097:blood / Q000302:isolation & purification; D011312:Pressure; D011487:Protein Conformation; D013950:Thymus Gland / Q000032:analysis
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006657:Histones