Molecular Characterization of a Bovine Growth Hormone Aggregate Species
Equilibrium denaturation studies of bovine growth hormone (bGH) have identified the existence of four distinct species: native, unfolded, a stable folding intermediate, and an associated folding intermediate. This associated intermediate, also referred to as an aggregate, is populated under conditions of partial denaturation and elevated protein concentrations. It has also been demonstrated that a region of bGH, spanning amino acid residues 96-133, helps to stabilize the aggregate. These findings indicate that bGH aggregation, and bGH(96-133) interactions with partially denatured bGH are noncovalent processes resulting from well-defined intermolecular interactions. This study attempts to characterize the complex formed between bGH(96-133) and partially denatured bGH at the molecular level. To accomplish this, I attempted to chemically crosslink partially denatured bGH and bGH(96-133) that was selectively tagged with a photolabile crosslinking reagent. In addition the photolabile derivatives were crosslinked with underivatized bGH(96-133) under conditions that promote peptide aggregation. It was found that the photolabile derivatives could be crosslinked to bGH and the bGH fragment, but that these products were produced in low yield. Further investigations showed that S-p-phenacyl-[Cys1l7]bGH(96-133) forms a complex with partially denatured bGH, and that this complex probably mimics the tertiary structure of bGH. Complex formation with S-p-phenacyl-[Cys110]- and -[Cys124]bGH(96-133) was inhibited both by steric hindrance resulting from introduction of the crosslinker, as well as decreased alpha-helical content as compared with the wild-type peptide.