Conformational effects of organic solvents on histone complexes.

Changes in the conformations of H3-H4, H2A-H2B, and the core histone complex brought about by the addition of organic solvents have been examined by circular dichroism spectroscopy. All three complexes assume increased alpha helicity with increasing amounts of the organic solvent. An amount of secondary structure equal to that obtained in phosphate-buffered 2 M NaCl solution can be induced in low-salt solutions of the complexes by the addition of 40-50% ethylene glycol, 50% glycerol, or approximately 2% hexafluoro-2-propanol. H3-H4 was found to be somewhat more flexible than H2A-H2B in its response to changes in solvent polarity. Upon being heated, H3-H4 and the core histone complex both undergo irreversible alpha leads to beta transitions in 50% ethylene glycol under low-salt conditions, while H2A-H2B undergoes an essentially reversible alpha leads to beta random-coil transition under the same conditions. These results are discussed in terms of the dynamics of the nucleosome particle.

Study of conformational states and reversibility of histone complexes.

The core histone complex (H3:H4:H2A:H2B)2 and products of dissociation, the H2A:H2B: dimer and the H3:H4 tetramer, were isolated from chicken erythrocyte chromatin by several literature methods as well as gel filtration on Bio-Gel A15m at various salt concentrations. The conformational and oligomeric characteristic of these histone complexes were compared to analogous histone complexes prepared by renaturation of individually acid-extracted histones by circular dichroism (CD) and analytical gel filtration chromatography. The salt-extracted core histone complex (independent of method of preparation), the purified dissociation products, the H2A:H2B dimer, and the H3:H4 tetramer in 2 M NaCl, 10 mM sodium phosphate, 0.25 mM EDTA and 0.1 mM DTT, pH 7.0, have conformation which are identical, by the criteria of similar CD spectra, with complexes prepared from acid-extracted histones, Likewise, the salt-extracted complexes may be cycled through solvents of low ionic strength (10 mM sodium phosphate, pH 7.0 or 50 mM NaOAc, pH 5.0) or 1 mM HCl and returned to 2.0 M NaCl, 10 mM sodium phosphate, 0.25 mM EDTA, and 0.1 mM DTT, pH 7.0, in a completely reversible manner. Thus it would appear that acid-denatured histones are capable of being fully renatured to yield native-like complexes.

Conformational changes in the H3 . H4 histone complex. Serological and circular dichroism studies.

The H3.H4 complex has been extracted from calf thymus chromatin in either 0.05 M NaOAc, pH 5.0, or in 2.0 M KCl, 0.1 M KPO4, pH 6.7, and both forms have been shown to consist solely of histones H3 and H4 when examined on sodium dodecyl sulfate-polyacrylamide gels. Serological and circular dichroism analyses indicate the existence of structural differences between the two forms. Dilution of the high salt form of the complex into low salt produces a time-dependent conformational change which is related to reduction in the alpha helix content of the complex and which exposes antigenic sites on the complex. The existence of multiple forms of the H3.H4 complex may be related to the dynamic equilibrium of nucleosome structure in vivo.

Conformational studies of the synthetic precursor-specific region of preproparathyroid hormone.

the secondary structure of a synthetic peptide representing the NH2-terminal, precursor-specific extension sequence of preproparathyroid hormone was studied. NH2-terminal extensions, or leader sequences, may serve a critical role in determining and facilitating the cellular secretion of proteins. These precursor regions, including the synthetic hormonal fragment studied, share common features of amino acid sequence and also may be similar in secondary structure. The secondary structure of the synthetic precursor peptide was predicted as described [Chou, P. Y. & Fasman, G. D. (1978) Adv. Enzymol. 47, 45-148]. The secondary structure was derived from circular dichroism spectra in both an aqueous buffer at physiological pH and in a nonpolar solvent selected to approximate the intramembranous environment. Two highly structured conformations were observed. In the aqueous buffer the secondary structure was 27% alpha-helix, 43% beta-sheet, and 30% random coil. In the nonpolar solvent the secondary structure was 46% alpha-helix, 0% beta-sheet, and 54% random coil. These findings correlated well with the two highest-probability structures predicted from the amino acid sequence. Both the relatively high content of secondary structure in a peptide of this size (30 amino acids) and the conformational transition observed in changing from aqueous to nonpolar environments may reflect structural properties critical to the physiological function of NH2-terminal extension sequences, and both are consistent with current theories regarding the role of precursor regions in the intracellular transport and secretion of proteins.

The thermodynamics of nucleotide binding to proteins.

Models describing the interaction of a small molecule with a protein are typically couched in terms of the stoichiometry, cooperativity, and binding free-energy change. These parameters are readily available from equilibrium dialysis experiments (or appropriate variations). With the recent advent of extremely sensitive calorimeters, it is possible to obtain thermal data for the binding reaction and, thus, the entire set of thermodynamic parameters, delta G', delta H', delta S', delta C', become readily available. This review is limited to the binding of nucleotides and nucleotide analogs to proteins for which complete thermal data are available. While the majority of such systems have been characterized by calorimetry, we have not excluded, per se, van't Hoff enthalpy determinations. The systems we have considered include, but are not limited to, thymidylate synthetase, phosphorylase, several dehydrogenases, aldolase, glutamine synthetase, hemoglobin, asparate transcarbamylase, and ribonuclease. A variety of forces contribute to the total free-energy change upon ligand binding. These forces include ionic, van der Waals, hydrogen bond, and hydrophobic. In several cases, properly designed experiments have allowed a partial resolution of the individual contributions of these various forces. Variation of easily accessible conditions such as temperature, pH, ionic strength, or solvent third component produce changes in the set of thermodynamic parameters which lead to the resolution of the forces. The generality of heat effects makes this method very useful for studying the involvement of protons in binding reactions. The variation in the magnitude and direction (release or uptake) of the proton flux is readily studied by determining the apparent heat of reaction at constant pH, ionic strength, and temperature in two or more buffers of differing heat of ionization. This application has been exploited in several cases and is examined in great detail. An overview of the results in these systems to date suggests that several trends observed in the thermodynamic parameters need to be confirmed by further experimentation and, if they hold, an appropriate theoretical basis must be developed to aid in their interpretation.