Kinetic analysis of the folding of human growth hormone. Influence of disulfide bonds.
We report the results of a stopped-flow kinetic evaluation of the folding of human growth hormone (hGH). The results are compared with those obtained for a disulfide-modified analog in which the four cysteine residues have been reduced and alkylated to form tetra-S-carbamidomethylated hGH in order to elucidate the role of disulfide bonds in the folding reaction. Multiple detection techniques were applied to monitor both refolding and unfolding processes initiated by guanidine hydrochloride concentration jumps. Using far-UV circular dichroism (CD) detection to monitor folding of hGH, we find that 70% of the secondary structure forms in a burst phase occurring within the stopped-flow dead time. Two slower phases were identified in the observable portion of the CD signal. Multiple kinetic phases were resolved when folding was monitored by intrinsic tryptophan fluorescence or near-UV absorbance as probes of tertiary structure, and the number of time constants required to fit the data depended on the hGH concentration and nature of the denaturant jump. The associated amplitudes also displayed strong dependence on the final denaturant concentration. Results obtained from the tetra-S-carbamidomethylated hGH studies demonstrate that the folding reactions of hGH are remarkably similar in the presence and absence of the disulfide bonds. Disulfide bond reduction in hGH is proposed to affect folding primarily by increasing the population of self-associated intermediate states in the folding pathway.
Youngman
K M
KM
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Spencer
D B
DB
Brems
D N
DN
DeFelippis
M R
MR
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D013006:Growth Hormone / Q000031:analogs & derivatives / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D004220:Disulfides; D006146:Guanidines; D011994:Recombinant Proteins; D013006:Growth Hormone; D019791:Guanidine