Infrared spectroscopy is an unusual method for studying conformational changes of proteins. Nevertheless, the method offers some advantages, such as the possibility to measure small amounts of samples in different states. We can obtain information about intermolecular interactions, and it is particularly sensitive in characterizing hydrogen bonds, which are among the leading forces that stabilize proteins.
The process of denaturation is very often coupled with protein aggregation. In this work, we followed the changes in the secondary structure of three model proteins under temperature and chemical denaturation conditions with sodium dodecyl sulfate.
We used difference spectroscopy to detect conformational changes of the proteins. By integrating peaks in difference spectra, we predicted denaturation temperatures of proteins and changes in the amount of secondary structures. We showed that we can detect changes in transition temperatures with FTIR when small amounts of denaturant are present. Using decomposition and peak fitting, we successfully predicted the amount of secondary structures in the native form and calculated the temperature dependence of the secondary structure content of α-helices and β-aggregates for hemoglobin.
Hemoglobin and conalbumin experienced aggregation throughout the working concentration range of FTIR spectroscopy. The presence of dodecyl sulfate resulted in slow aggregation of the proteins, which we detected with differential spectroscopy. Characterization of spectra with high concentrations of dodecyl sulfate was not possible due to saturation of the protein spectra.
Insulin at pH 3 showed no significant changes in secondary structure besides the weakening of hydrogen bonds at higher temperature measurements. These data are consistent with previous studies of insulin showing that fibrillation begins only after an initial lag phase.
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