Site-directed mutagenesis to probe protein folding: evidence that the formation and aggregation of a bovine growth hormone folding intermediate are dissociable processes.
Bovine growth hormone (bGH) forms a stable folding intermediate that aggregates at elevated concentrations (greater than 10 microM). Thermodynamic and kinetic studies have shown that the formation of this bGH folding intermediate and its aggregation are separate processes, implying that selective modifications of bGH can lead to their independent modulation. In addition, a bGH region that includes amino acid residues 109-133 appears to be directly involved in this aggregation process. Human growth hormone (hGH), which is unable to aggregate via this mechanism, differs from the bovine primary sequence at eight positions within this protein region. We have characterized the folding of a bGH analogue that contains the hGH sequence between amino acid residues 109-133 (8H-bGH) at low and high concentrations. The equilibrium folding characteristics of bGH and 8H-bGH are similar when monitored at low protein concentrations (less than or equal to 2 microM). The wild-type and analogue proteins have equivalent denaturation midpoints when equilibrium unfolding is monitored by the use of far-UV circular dichroism, second-derivative UV, or fluorescence. In addition, the enhanced fluorescence that is associated with the formation of the bGH monomeric folding intermediate (Havel, H. A., et al. (1988) Biochim. Biophys. Acta 955, 154-163) is observed for 8H-bGH under similar conditions. In contrast, partial denaturation of 8H-bGH at higher concentrations (greater than 2 microM) leads to significantly less aggregation than is observed for bGH. This result is obtained from near-UV CD spectroscopy, kinetic folding, size-exclusion chromatography, and dynamic light-scattering data.(ABSTRACT TRUNCATED AT 250 WORDS)
Lehrman
S R
SR
Control Development and Molecular Biology Research, Upjohn Company, Kalamazoo, Michigan 49001.
Tuls
J L
JL
Havel
H A
HA
Haskell
R J
RJ
Putnam
S D
SD
Tomich
C S
CS
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D004926:Escherichia coli / Q000235:genetics; D013006:Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics
D011994:Recombinant Proteins; D013006:Growth Hormone