Investigating the Structural Change in Protein Aqueous Solution Using Temperature-Dependent Near-Infrared Spectroscopy and Continuous Wavelet Transform

The circulatory protein, human serum albumin (HSA), is widely used as a model protein for the study of protein structure. In this work, the structures of human serum albumin in aqueous solutions are studied using temperature-dependent near-infrared (NIR) spectroscopy with the aid of continuous wavelet transform (CWT). Near-infrared spectra of human serum albumin solutions with different concentrations were measured over a temperature range of 30-85 degrees C. Then, continuous wavelet transform was performed on the spectra to enhance the resolution. As a result of the resolution enhancement, spectral bands around 4361, 4521, 4600 and 4260 cm(-1) were extracted from the overlapping low-resolution signals. The four bands can be assigned to the protein structures of a-helix, b-sheet, an intermediate state and side chains, respectively. The variations in intensity of the bands around 4361 and 4521 cm(-1) with temperature show that the increase of temperature leads to the loss of a-helical structure but the formation of beta-sheet, and the denaturation temperature of human serum albumin is about 55 degrees C. The variation of the band around 4600 cm(-1) indicates that the temperature-induced unfolding process of human serum albumin occurs through a stable intermediate state, and a significant change in the micro-environment of the side chains about 63 degrees C is observed from the variation of the band around 4260 cm(-1). On the other hand, the transformed spectra in the region of 8000-5600 cm(-1) provide an explicit evidence for the structural changes of water during the process of protein denaturation, and the unfolding process of HSA can be reflected by these changes.