Protein structure in vivo is generally different from that of “frozen” crystal structure. In some cases, such a discrepancy can be so large that it leads to the biological activity of the protein being totally different from that associated to the crystal structure. Therefore, it is imperative to develop physical methods to solve, at least partially the dynamical protein structure under physiological condition.　
  Temperature-jump time-resolved mid-IR transient absorbance difference spectroscopy is one of such a method having the capability of high temporal resolution without damaging the sample when the experimental condition is appropriately controlled. It employs a heating laser to induce a dynamical protein structural change, and uses another mid-IR laser to detect the protein secondary structure in the fingerprint region. Yuxiang Weng’s group (Laboratory of Soft Matter Physics, Institute of Physics, CAS) has home-built a temperature-jump time-resolved mid-IR transient absorbance difference spectrometer based on domestic technique of carbon monoxide laser as the IR probe, and they have successfully used this apparatus to investigate the protein dynamic structure (Biophysical Journal, 93, 2756-2766, 2007).
  Recently, through interdisciplinary collaboration, Weng’s group and  Chih-chen Wang’s group (National Laboratory of Biomacromolecules, Institute of Biophysics, CAS), together with Dr. Guoping Ren (Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Michigan Michigan), investigated the thermal stability of disulfide bond isomerase, a homodimeric DsbC, and its thermal-dependent oxidase activity, solved the puzzle regarding the abnormal activity of disulfide bond isomerase, i.e., DsbC dimer being a reductase can even participate in the oxidizing reaction pathway. The result was published in Biophysical Journal, 97, 2811–2819, 2009. This work is an interdisciplinary research combining physics, biochemistry and molecular biology. Disulfide bond isomerase locates in the periplasm of the bacteria, chained in the reduction pathway as reductase responsible for opening of the mismatched disulfide bonds. Crystal structure shows that it consists of two DsbC monomers linked  via 9 pairs of hydrogen bonds at the dimeric interface. In the periplasm, there exists another parallel pathway consisting of monomeric oxidase (DsbA and DsbB), which is responsible for oxidizing the thiol groups of the substrate protein to the disulfide bond. These two pathways are separated and no cross-talk is expected. However very recently it is found that DsbC dimer can contribute to the oxidizing pathway by acquiring an oxidase activity to a certain extent, which remains as a puzzle. For disulfide bond isomerase would be incapable of participating the oxidation reaction due to the steric hindrance based on its dimeric crystal structure. 
  Steady-state Fourier Transformation IR spectra show that at physiological condition and a relatively lower temperature, DsbC dimer tends to undergo conformational changes. T-jump time-resolved IR spectra further reveals that the hydrogen bonds at the dimeric interface break within a period of about 30 ns when subjected to the heat shock. Meanwhile the corresponding time-resolved IR difference spectra confirm the formation of the DsbC monomers, indicating a thermal-induced dimeric dissociation process occurs. Later the thermal-induced dissociation of the DsbC dimer was verified via biochemical methods, and the thermal-dependent oxidase activity of DsbC both in vivo and in vitro were determined. The results confirmed that DsbC dimer does have abnormal activity as an oxidase, and this oxidase activity increase with the increases in temperature. Thus the thermal-induced dissociation of DsbC dimmer enables it has an unexpected oxidase activity due to the formation of monomer, which accounts the puzzle at the molecular level.
  This work was supported by the National key fundamental research program, the China National Science Foundation and Chinese Academy of Sciences Innovative Project.　 

Schematic diagram for thermal-induced dissociation of disulfur bond isomerase (DsbC dimer). Experiments reveal that the hydrogen bonds linking the two monomers at the dimeric interface break within a period of about 30 ns when subjected to the heat shock, indicating a thermal-induced dimeric dissociation occurs.