Plasma serine proteinase inhibitors (serpins) exhibit major conformational changes and a large increase in conformational stability upon cleavage at their reactive sites.
Intact and proteolytically modified human serpins, alpha 1-proteinase inhibitor, antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor, were compared by circular dichroism, fluorescence spectroscopy, and resistance against unfolding by guanidine HCl. The modified proteins were prepared from the intact and active inhibitors by selective proteolytic cleavage in their reactive site loops and tested for complete loss of activity. Significant differences in the spectral properties between intact and modified inhibitors indicate that a major conformational rearrangement is triggered by the cleavage. This leads to a large increase in conformational stability as demonstrated by large shifts of the transition profiles recorded as a function of guanidine HCl concentration at 20 degrees C by circular dichroism at 220 nm. Intact inhibitors were unfolded in two steps of about equal size centered at 0.8-1.7 and 2.5-3.5 M concentrations of the denaturant, respectively. Under identical conditions modified inhibitors are completely stable, and their denaturation occurs only well above 4 M guanidine HCl in one or two steep transition steps. From the similarity of the spectral changes and shifts in transition profiles for all four serpins studied it is concluded that the conformational changes and stabilization triggered by the modification hit is an important common mechanistic feature of this class of inhibitors. This is supported by the observation that ovalbumin, which is homologous with the serpins but apparently lacks inhibitory activity, exhibits neither spectral changes nor a significant change in stability upon proteolytic modification.
Bruch
M
M
Department of Biophysical Chemistry, University of Basel, Switzerland.
Weiss
V
V
Engel
J
J
D000465:Algorithms; D000990:Antithrombin III; D001665:Binding Sites; D001798:Blood Proteins; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D011480:Protease Inhibitors; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin
D001798:Blood Proteins; D006146:Guanidines; D011480:Protease Inhibitors; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D000990:Antithrombin III; D019791:Guanidine