A new spectroscopic approach to examining the role of disulfide bonds in the structure and unfolding of soybean trypsin inhibitor.
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 structural changes 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 secondary structure of soybean trypsin inhibitor (STI). The disulfide bonds are reduced with tris(2-carboxyethyl)phosphine (TCEP) at 40 degrees C, and the reduction process is probed in real-time using sulfur X-ray absorption spectroscopy. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies are used concurrently to determine the structural changes caused by reduction of the disulfide bonds. Results demonstrate a noncooperative reduction of the two disulfide bonds within 5 min, likely because they are located on the surface of the protein. The unfolding of STI lags behind; dramatic changes are not observed until 60-90 min after the reduction was initiated. The CD and FTIR spectra indicate a decrease in the amount of extended (hydrated) coil, suggesting that the STI structure slowly collapses after the disulfide bonds are reduced. Thus, although the disulfide bonds are not located near the active site of STI, they play a crucial role in stabilizing the protein structure, which is necessary to sustain enzymatic activity.
Tetenbaum
J
J
National Synchrotron Light Source, Brookhaven National Laboratory, Upton, New York 11973, USA.
Miller
L M
LM
D004220:Disulfides / Q000737:chemistry*; D004577:Electron Probe Microanalysis / Q000379:methods*; D015394:Molecular Structure; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry*
D004220:Disulfides; D014360:Trypsin Inhibitor, Kunitz Soybean