Two-Dimensional Attenuated Total Reflection/Infrared Correlation Spectroscopy of Adsorption-Induced and Concentration-Dependent Spectral Variations of β-Lactoglobulin in Aqueous Solutions

Abstract

This paper demonstrates the potential of generalized two-dimensional (2D) attenuated total reflection/infrared (ATR/IR) spectroscopy in studies of spectral variations in the amide I region of aqueous solutions of protein. Two examples of the 2D correlation analysis are discussed in this paper. The first is concerned with adsorption-dependent spectral changes of β-lactoglobulin (BLG) in solution. The second approach is dedicated to the concentration-dependent spectral changes. To generate the 2D correlation spectra, the original spectra have been subjected to pretreatment procedures consisting of ATR correction, subtraction of the spectrum of the buffer solution, smoothing, and normalization over the concentration. The adsorption-dependent 2D study shows that the interaction between the crystal surface and the protein molecules can be monitored successfully by the synchronous and asynchronous correlation spectra. This interaction is characterized by pronounced intensity changes at frequencies assigned to β-sheet elements buried in the hydrophobic core of the molecule. The concentration-dependent 2D correlation maps, which develop 10 times more intense features than the adsorption-dependent 2D maps, are concerned with changes in various secondary structure elements located in the hydrophilic parts facing the solvent. The present study has also aimed at expanding generalized 2D correlation spectroscopy to quantitative utilization. The quantitative analysis of the 2D maps reveals that the intensity changes observed for the series of aqueous solutions of BLG with different concentrations are predominantly composed of concentration-induced secondary structure changes even in the presence of the adsorption of protein molecules to the ATR crystal.

Multivariate Estimation between Mid and Near-Infrared Spectra of Hexafluoroisopropanol-Water Mixtures

“…First, an ATR correction was performed to each individual IR spectrum. 12 Then, subtraction of the contribution of vapor water from each spectrum was performed.…”

Pressure-induced Structural and Hydration Changes of Proteins in Aqueous Solutions

“…The obtained spectra were baseline corrected in the region of the amide I band. 14,15 Curve fitting with Gaussian functions was performed using Origin software. PCA was performed with the treated spectra after mean-centering using the Unscrambler 5.0 program.…”

2 D Correlation Spectroscopy in Vibrational Spectroscopy

“…4,29,37,43,50,53 -57,64,65,68 -71 Thus, numerous 2D correlation spectroscopy studies have been reported for concentration-or composition-dependent spectral variations of proteins, 29,43,50,57,70 polymers 37,53,54,56,64,65,68,69,71 and multicomponent mixtures. 4,55 For example, systematic studies of polymer blends and copolymers exhibiting specific interactions of components using 2D IR, 2D NIR, 2D Raman and hetero-correlation analysis have been reported.…”

“…Czarnik-Matusewicz et al 43 reported, based on the 2D IR study on the amide I region, that the concentration change in the BLG solutions induces environmental changes in BLG, leading to the association of the protein. According to the authors, 43 exposed residues of BLG may be responsible for these concentration-dependent structural changes.…”

“…According to the authors, 43 exposed residues of BLG may be responsible for these concentration-dependent structural changes. The intensity of the autopeaks in the synchronous 2D IR and 2D Raman spectra in amide III region (Figures 45 and 46) indicates that the random coil structure varies most significantly with the formation of association.…”