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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 Parathyroid hormone (PTH), like other secreted proteins (1-14), is initially biosynthesized in a larger precursor form, preproparathyroid hormone (preproPTH), which contains an NH2-terminal extension, or leader sequence (15). Although the biological role of precursor regions in the biosynthesis, posttranslational modification, and intracellular transport of secreted proteins remains to be fully elucidated, it is thought that leader sequences promote the initial steps in protein secretion: the interaction of nascent peptide chains with intracellular membranes and the subsequent entry of protein into the cisternae of the rough endoplasmic reticulum (RER) (16)(17)(18)(19)(20). Although the amino acid sequences of precursor regions from various preproteins differ widely, the NH2-terminal extensions share common structural features. They are similar in length and contain a central highly hydrophobic sequence (1-15, 21, 22). Furthermore, predictions of the secondary structure of a number of these regions have demonstrated a high degree of conformational similarity among the sequences, despite differences in the physiological function and the species of origin of the proteins examined (23-26). Additional evidence for conformational similarity among leader sequences is suggested by the finding that related preproteins from one species may be accurately cleaved to their mature forms by microsomal membranes derived from another species (27, 28).At present, quantities of native precursor-specific peptides sufficient to permit conformational studies are not available. In vivo, leader sequences are rapidly removed from nascent peptide chains before protein secretion, perhaps before biosynthesis of the entire preprotein is complete (29, 30), and thus far have not been subsequently identified and isolated (31-34). In vitro, the entire preprotein can be biosynthesized using cell-free translational systems. However, it is likely that the conformation adopted by the precursor-specific region, when it is contiguous with the complete mature protein sequence, may differ from the conformation it adopts early in protein biosynthesis. Finally, it is not yet practical to obtain large quantities of leader-sequence peptides by exposing preproteins to microsomal membranes in vitro.To study the conformation of an isolated precursor sequence, we used a chemically synthesized peptide representing the NH2-terminal extension of preproPTH (35). The 30-amino acid, single-chain peptide, [D-Tyr+...

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.

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