A study of the self-association and conformation of histone F2a2

Abstract

Histone F2a2 has been studied by sedimentation velocity, sedimentation equilibrium, viscosity, and optical rotatory dispersion (ORD) techniques. Measurements were performed as a function of temperature, and NaCl and GuHCl concentration in 0.005 M phosphate buffer at pH 7.0. F2a2 appears to be more sensitive to phosphate than previously shown for histones F2al or F2b. Under these conditions F2a2 contains as much as 11% helical structure and appears to exist as a monomer at least at low protein concentrations. The addition of salt causes considerable aggregation. In 0.15 M NaCl the sedimentation coeffi.cient represents an almost seven-fold increase over that in buffer. The self-association of F2a2 in 0.15 M NaCl has also been studied by low speed equilibrium experiments. The concentration dependence of the apparent weight average molecular weight, M[subscript wa], was indicative of a nonideal self-associating system. Several model systems were tested and the data was best described by a monomer-n-mer with an extremely large equilibrium constant and a relatively small nonideality term. The lower limit was taken to be 23 that of a monomer-12 mer with K = 4 X 10²³ and BM₁= 0.0015. A slight increase in the ordered structure results from binding of the salt, histone-histone interaction or both. There appears to be only about a 2.5% increase in helical structure in going from an apparent monomer in 0.005 M phosphate to an apparent 12 mer in 0.15 M NaCl. The aggregation and conformation of F2a2 is also dependent on temperature. Higher temperatures result in an increase in ordered structure as well as degree of association. The transition from 10° to 40°C involves the formation of as much as 6% additional α-helix. The F2a2 aggregate is very resistant to GuHCl. At a concentration of 0.75 g/100 ml 6 M GuHCl is required for complete dissociation and denaturation. Only about half of the histone F2a2 polypeptide chain is probably involved in these conformational and associative changes, while the rest may be free to bind to DNA. The strength of these interactions suggests that they are indeed important in the maintenance and function of chromatin.