Graduate Thesis Or Dissertation
 

Physical properties of calf thymus H1 histones : conformational changes and interactions with two nonhistone chromosomal proteins

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  • This work represents the first study of the conformational changes in histone H1 subfractions as well as the interactions of these subfractions with two nonhistone chromosomal (NHC) proteins. Calf thymus Hi was fractionated by the method of Kincade and Cole (1966a) using a very shallow guanidinium chloride gradient. A possible new Hi subfraction, about 5-8% of the whole H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission, the circular dichroism (CD) of the peptide bond, and the tyrosine absorbtion. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD, with no further time dependence. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine (at, or near, residue 72), which becomes buried in the folded form. The folding of subfraction 3a is approximately twice as sensitive to salt as the other major subfractions. Upon folding, approximately 13% of the residues of subfractions lb and 2 form α and β structure; 3a and 3b have approximately 16% of the residues in α and β structures. There is no evidence for interactions between the subfractions, as measured by fluorescence anisotropy and sedimentation equilibrium. In salt free solutions, each of the four major subfractions shows very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity and involves the formation of both α and β structure in a manner similar to that of the salt induced state. The interactions of the NHC proteins HMGI and HMG2 with the four major HI subfractions were studied using fluorescence anisotropy of the tryptophan emission, sedimentation equilibrium, sedimentation velocity, and CD. HMG1 and HMG2 interact with the various subfractions of calf thymus H1 with a high degree of specificity. Subfractions lb and 2 interact very strongly with HMG1 to form heterodimers. in contrast, subfractions 3a and 3b interact much more weakly. The formation of the two strong complexes involves little or no change in α-helical or β-sheet content. Also, the Hl(2).HMG1 complex has an overall tertiary structure that is more globular than that of the H1(2) molecule alone. HMG2 interacts with 3a and 3b but shows no detectible complexing with la and 2.
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