A Study of Urea-dependent Denaturation of beta-Lactoglobulin by Principal Component Analysis and Two-dimensional Correlation Spectroscopy

The water-urea-beta-lactoglobulin interaction was studied by means of principal component analysis (PCA) and two-dimensional correlation spectroscopy applied to the urea concentration-dependent FTIR spectra of aqueous urea-protein solutions. The two nu(C=O) and nu(as)(CN) bands coming from urea's absorbance, instead of the amide bands arising from protein, were employed in the analysis. To get a precise view of the changes induced by the urea concentration-control led unfolding process, the absorbance variations developed in the ternary water-urea-protein system were compared with those observed in a binary water-urea system [Y.M. Jung et al, J. Phys. Chem. B 2004, 108, 13008]. The comparative studies enabled to detect apparent differences between the absorbance changes caused solely by urea's concentration increase and by the urea-dependent unfolding process. Urea's ability to unfold protein was discussed in context of the indirect and the direct mechanism depending on urea's concentration. It was shown that both mechanisms are relevant, that is, the indirect for solutions below 3 M and the direct for solutions above 3 M concentration. The character of the mechanism is strictly correlated with the association level of urea molecules.X113337sciescopu

Characterization of concentration-dependent infrared spectral variations of urea aqueous solutions by principal component analysis and two-dimensional correlation spectroscopy

The association behavior of urea as a function of urea concentration was elucidated by applying principal component analysis (PCA) and two-dimensional (2D) correlation spectroscopy to concentration-dependent FTIR spectra of aqueous urea solutions. The first principal component revealed changes that occurred during the initial increase in urea concentration, which led to the formation of dimers. The second principal component gave information on changes involving the formation of higher aggregates at higher urea concentration. By applying 2D correlation analysis to two sets of FTIR spectra collected above and below 2 M of urea, which were divided on the basis of the PCA, further details on the association process were extracted. Synchronous and asynchronous 2D correlation spectra constructed from the-first group of spectra (urea concentration up to 2 M) identified intensity changes due to the formation of ribbon and chain dimers, while the 2D correlation analysis indicated that for urea concentrations above 2 M, urea undergoes further chain polymerization.X112828sciescopu

Two-dimensional correlation analysis of Raman optical activity data on the α-helix-to-β-sheet transition in poly(L-lysine)

Raman optical activity (ROA) has evolved into an incisive probe of structure and conformational transitions in polypeptides and proteins revealing many signal patterns characteristic of specific secondary structural elements. In order to further facilitate analysis of ROA spectral intensity variations, two-dimensional correlation methods are applied to ROA and Raman spectra monitoring the α-helix-to-β-sheet transition in poly(L-lysine) as a function of temperature. Pretreatment of data using background subtraction, normalization and gentle smoothing is essential for the successful generation of 2D ROA correlations, 2D Raman correlations and 2D Raman/ROA heterocorrelations. The pseudoscalar nature of ROA spectra results in detailed 2D correlation analyses providing extensive interpretation of spectral intensity variations. Synchronous plots indicate band assignments consistent with established assignments in poly(L-lysine) together with possible new assignments. Corresponding asynchronous plots probe the temporal sequence of the conformational transition indicating distinct temporal phases while monitoring aggregation through a small amount of β-structure present at the start of the experiment ahead of α-helix unfolding. This study demonstrates the potential of 2D correlation analysis as a valuable technique for the extraction of detailed information about aggregation and conformational transitions in polypeptides and proteins from associated ROA and Raman spectra. Results indicate that aggregation of poly(L-lysine) monomers precedes intramolecular conversion of α-helix to β-sheet, which is then followed by fibril formation

Characterization of transition temperatures of a Langmuir-Blodgett film of poly(tert-butyl methacrylate) by two-dimensional correlation spectroscopy and principal component analysis

External reflection FT-IR spectra of a Langmuir-Blodgett (LB) film of poly(tert-butyl methacrylate) (PtBMA) were measured at temperatures ranging from 26 to 136??C. The glass transition temperature (Tg) was determined from a two-dimensional (2D) mapping of the first derivative spectra of absorbance values against temperature over the wavenumber range 1100-1300 cm-1, which contains spectral features that are very sensitive to conformational changes. This mapping provides a surprisingly simple and direct method for detecting the value of Tg. The glass transition temperature determined from the 2D map was approximately 84??C. Another transition at 103??C, corresponding to the glass transition temperature of bulk PtBMA, was also detected from the 2D map. Principal component analysis (PCA) was employed to analyze the temperature-dependent FT-IR spectra. The glass transition temperatures (80??C; 100??C) of the PtBMA LB film determined by the score plot of PCA are consistent with those determined by the 2D map. Additionally, the loading vectors of PCA were found to give valuable insight into the molecular-level phenomena associated with the glass transition process. To gain more details about the polymer chain mobility, two-dimensional (2D) correlation analysis was performed on two sets of FT-IR spectra collected above and below Tg. In the synchronous 2D correlation spectrum obtained below the glass transition temperature (26-66??C), the observation that the strongest intensity change occurs at 1137 cm-1 indicates that the reorganization of the bending mode of the bbC-C-O and bbC-C=O bonds connected to the backbone (bb) chain and coupled to the C-O stretching mode of the tert-butoxy group is potentially the mechanism underlying the ??-transition. This result is in good agreement with the presence of a transition (??-transition) at approximately 43??C obtained from the band around at 1137 cm-1 in the 2D mapping data.close303

Two Novel Spectroscopic Techniques: 2D ROA and 2D Raman/ROA

No abstract available

Two Novel Spectroscopic Techniques: 2D ROA and 2D Raman/ROA

No abstract available