In the preceding paper [Havel, H. A., Kauffman, E. W., Plaisted, S. M., & Brems, D. N. (1986) Biochemistry (preceding paper in this issue)], an associated intermediate was shown to be highly populated during the equilibrium denaturation of bovine growth hormone. In this paper, we describe its partial characterization and propose a mechanism for association. The associated equilibrium intermediate is populated under conditions that induce partial denaturation and at protein concentrations greater than 0.2 mg/mL. The remaining nativelike helical structure present in the partially denatured species is implicated in the mechanism of association as demonstrated by similar concentration dependencies and thermal stabilities of the helix and the associated equilibrium intermediate. Furthermore, it is suggested that a putative amphiphilic helix from residues 110-127 plays a critical role in the association as demonstrated by a diminution of the associated equilibrium intermediate when mixed with the peptide fragment 96-133. A model is proposed to account for these results in which partial denaturation exposes the segment of the protein corresponding to the hydrophobic face of the putative amphiphilic helix 110-127. This metastable form is the species from which association occurs. Association is stabilized by the hydrophobic interactions resulting from intermolecular packing of the lipophilic faces of the helices. The implications of these results to protein folding studies are described.

Previous investigations have shown that bovine growth hormone (bGH, somatotropin) unfolds through a reversible multistate process with at least one stable equilibrium intermediate. In extending our knowledge of the folding process for bGH, we demonstrate that a self-associated form of partially denatured bGH is formed during equilibrium unfolding experiments. The self-associated species has been identified by hydrodynamic measurements (size exclusion high-performance liquid chromatography and static and dynamic light scattering) and by measurements of the bGH concentration dependence of aromatic amino acid spectral properties (fluorescence, second-derivative absorption, and circular dichroism). The apparent maximum concentration for self-association occurs when bGH is partially denatured, i.e., at 3.7 M guanidine hydrochloride or 8.5 M urea, and its formation is reversible. Some of the properties of the self-associated species include quenched tryptophan fluorescence, increased tryptophan circular dichroism intensity at 300 nm, polar tryptophan environment, and a weight-average radius of about 5 nm. The self-association of bGH is mediated by specific intermolecular interactions with little increase in molecular size occurring above the saturation level of 4 mg/mL bGH. These phenomena have important implications for the design and interpretation of folding experiments in vitro and may have physiological consequences.

The peptide 109-133 was isolated from bovine growth hormone (bGH) and studied for helix formation in aqueous solutions. This fragment was shown to contain helical structure by far-ultraviolet circular dichroism in aqueous solutions. The amount of helix was dependent on pH and peptide concentration. The peptide has maximum helicity between pH 4 and 5 and at high peptide concentration. Under these conditions for maximal helix population, this fragment is approximately 100% helical. Secondary structure predictions suggest that residues 110-127 have a strong propensity to form an amphipathic helix. We have also studied a related peptide, 96-133, and show by gel filtration that it undergoes an increase in molecular weight that directly correlates with a coil to helix transition. A comparison of the helical content of 96-133 to 109-133 and circular dichroism studies of peptide 96-112 suggest that the helix of 96-133 is limited to the 109-133 region. Current models for alpha-helix formation predict that peptides the size of 109-133 should not contain measurable helicity in aqueous solutions. Our studies show that the unusual stability of helix 109-133 is due to electrostatic interactions and probable intermolecular packing between hydrophobic faces of the amphipathic surfaces of the helices. The implications of helix formation in these fragments to a framework model of protein folding for bGH are discussed.

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