Reversible denaturation of the soybean Kunitz trypsin inhibitor.
The soybean Kunitz trypsin inhibitor (SKTI) is a beta-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and renaturation of SKTI. Upon heating to 70 degrees C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 degrees C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 degrees C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 degrees C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered beta-sheet proteins.
Roychaudhuri
Robin
R
Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664, USA.
Sarath
Gautam
G
Zeece
Michael
M
Markwell
John
J
D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D004220:Disulfides; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry*; D014466:Ultraviolet Rays
D004220:Disulfides; D014360:Trypsin Inhibitor, Kunitz Soybean; D014357:Trypsin