| Abstract
| - Although it is well-known that disulfide bonds stabilize the secondary structure of many proteins,it is difficult to directly probe both disulfide bond formation/breakage and the resulting secondary structuralchanges during the course of the protein folding/unfolding process. In this work, we have used a new,real-time spectroscopic approach to examine how the reduction of two disulfide bonds affects the secondarystructure of soybean trypsin inhibitor (STI). The disulfide bonds are reduced with tris(2-carboxyethyl)phosphine (TCEP) at 40 °C, and the reduction process is probed in real-time using sulfur X-ray absorptionspectroscopy. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies are usedconcurrently to determine the structural changes caused by reduction of the disulfide bonds. Resultsdemonstrate a noncooperative reduction of the two disulfide bonds within 5 min, likely because they arelocated on the surface of the protein. The unfolding of STI lags behind; dramatic changes are not observeduntil 60−90 min after the reduction was initiated. The CD and FTIR spectra indicate a decrease in theamount of extended (hydrated) coil, suggesting that the STI structure slowly collapses after the disulfidebonds are reduced. Thus, although the disulfide bonds are not located near the active site of STI, theyplay a crucial role in stabilizing the protein structure, which is necessary to sustain enzymatic activity.
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