pH-induced conformational states of bovine growth hormone.
The folding behavior of bovine growth hormone (bGH) is examined by chemical and pH denaturation using several spectroscopic probes of protein secondary and tertiary structure. Partially denaturing concentrations of urea eliminate the native-state quenching of intrinsic tryptophan fluorescence, from the single protein tryptophan, but the fluorescence emission spectrum is not red-shifted like the unfolded state, and the protein retains substantial secondary structure. A neutral-to-acid pH shift also eliminates tryptophan quenching; however, the loss of quenching is not accompanied by an emission red-shift. In addition, the protein undergoes a pH-dependent UV absorbance transition; the changes in absorptivity have the same midpoint as the transition associated with the change in intrinsic tryptophan fluorescence. The magnitude of the absorption transition is similar to that observed previously for urea denaturation of the protein. In a similar fashion, a pH-dependent CD transition is also observed; however, the transition occurs at a higher pH. The behavior of the various optical probes indicates that the pH-induced conformational transition produces a highly populated species in which the microenvironment surrounding the single protein tryptophan residue resembles that observed during the urea-induced unfolding/refolding transition. The pH-induced changes in tertiary structure occur at a lower pH than the changes associated with a portion of the secondary structure. Proton NMR of the low-pH intermediate indicates that the three His and six Tyr resonances are indistinguishable from the unfolded state. The intermediate(s) observed by either chemical or pH-induced denaturation resemble(s) a molten globule state which contains significant secondary structure. The residual secondary structure present in the intermediate could be nonnative.(ABSTRACT TRUNCATED AT 250 WORDS)
Holzman
T F
TF
Pharmaceutical Discovery Research, Abbott Laboratories, Abbott Park, Illinois 60064.
Dougherty
J J
JJ
Brems
D N
DN
MacKenzie
N E
NE
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D013006:Growth Hormone; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays; D014508:Urea
D014508:Urea; D013006:Growth Hormone