Evidence for a self-associating equilibrium intermediate during folding of human growth hormone.
It has been previously shown, by equilibrium denaturation, that human growth hormone (hGH) folds by a cooperative two-state process. This is in contrast to the folding pathways of other nonhuman growth hormones that contain stable monomeric and multimeric equilibrium intermediates. We have reinvestigated the equilibrium denaturation of hGH at higher protein concentrations and found smooth transitions from the native to denatured state, but the calculated free energy for unfolding, delta G, decreases with increasing protein concentration. The effect of protein concentration on the delta G of unfolding is due to the presence of folding intermediates that have a tendency to self-associate. A correlation was found between the equilibrium denaturation data and the observation of precipitation that occurs upon refolding, suggesting that the presence of self-associated folding intermediates leads to precipitation. Direct evidence for the existence of a soluble, associated intermediate was obtained by dynamic light scattering (DLS) and equilibrium analytical ultracentrifugation. Peptide fragments from the third helix of either hGH or bovine growth hormone (bGH) were capable of inhibiting the formation of this aggregated species and prevent precipitation during refolding. The data show that the folding pathway of hGH is similar to that of nonhuman growth hormones except for differences in the tendency for intermediates to self-associate. These findings are relevant to the design and interpretation of equilibrium folding experiments, and may be important to understanding mechanistic details of protein folding and aggregation in vivo.
DeFelippis
M R
MR
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285.
Alter
L A
LA
Pekar
A H
AH
Havel
H A
HA
Brems
D N
DN
D002851:Chromatography, High Pressure Liquid; D013006:Growth Hormone / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008027:Light; D008970:Molecular Weight; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation
D006146:Guanidines; D011994:Recombinant Proteins; D013006:Growth Hormone; D019791:Guanidine