Differences between the Pressure- and Temperature-Induced Denaturation and Aggregation of β-Lactoglobulin A, B, and AB Monitored by FT-IR Spectroscopy and Small-Angle X-ray Scattering

Abstract

We examined the temperature- and pressure-induced unfolding and aggregation of beta-lactoglobulin (beta-Lg) and its genetic variants A and B up to temperatures of 90 degrees C in the pressure range from 1 bar to 10 kbar. To achieve information simultaneously on the secondary, tertiary, and quaternary structures, we have applied Synchrotron small-angle X-ray diffraction and Fourier transform infrared spectroscopy. Upon heating a beta-Lg solution at pH 7.0, the radius of gyration Rg first decreases, indicating a partial dissociation of the dimer into the monomers, the secondary structures remaining essentially unchanged. Above 50 degrees C, the infrared spectroscopy data reveal a decrease in intramolecular beta-sheet and alpha-helical structures, whereas the contribution of disordered structures increases. Within the temperature range from 50 to 60 degrees C, the appearance of the pair distance distribution function is not altered significantly, whereas the amount of defined secondary structures declines approximately by 10%. Above 60 degrees C the aggregation process of 1% beta-Lg solutions is clearly detectable by the increase in Rg and intermolecular beta-sheet content. The irreversible aggregation is due to intermolecular S-H/S-S interchange reactions and hydrophobic interactions. Upon pressurization at room temperature, the equilibrium between monomers and dimers is also shifted and dissociation of dimers is induced. At pressures of approximately 1300 bar, the amount of beta-sheet and alpha-helical structures decreases and the content of disordered structures increases, indicating the beginning unfolding of the protein which enables aggregation. Contrary to the thermal denaturation process, intermolecular beta-sheet formation is of less importance in pressure-induced protein aggregation and gelation. The spatial extent of the resulting protein clusters is time- and concentration-dependent. The aggregation of a 1% (w/w) solution of A, B, and the mixture AB results in the formation of at least octameric units as can be deduced from the radius of gyration of about 36 A. No differences in the pressure stability of the different genetic variants of beta-Lg are detectable in our FT-IR and SAXS experiments. Even application of higher pressures (up to 10 kbar) does not result in complete unfolding of all beta-Lg variants.

Hydration and Packing Effects on Prion Folding and β-Sheet Conversion

“…Fourier transform infrared spectroscopy has been widely used to probe transitions in secondary conformation of proteins (6,17,31,41), and the use of a diamond anvil cell provides access to secondary structure components of proteins also upon pressurization (31,33).…”

“…A diamond anvil cell (High Pressure Diamond Optics Inc.) was used for the measurements under pressure. The samples were mixed with powdered ␣-quartz in a stainless-steel compartment (0.5 mm in diameter, 0.05 mm deep) (31,33), and changes in pressure were quantified by the shift of the quartz phonon band at ϳ798 cm Ϫ1 (36). The pressure studies were carried out between 1 bar and ϳ10 kbar.…”

Hydrogen Peroxide-induced Structural Alterations of RNase A

“…1) was correlated to the formation of aggregates of this protein. Previous IR studies have shown (48,50,51) that densely packed intermolecular ␤-aggregates give rise to highly characteristic infrared bands. Particularly, a so-called "␤-aggregation band" at 1615 cm Ϫ1 is known to be typical for the presence of these aggregates.…”

Islet amyloid polypeptide and high hydrostatic pressure: towards an understanding of the fibrillization process

“…On the other hand, the CD spectrum of the pressurized IAPP sample looks different, with a similar minimum but shifted to about 222 nm, however the overall CD ellipticity has decreased drastically, most likely due to precipitation of the IAPP aggregate (as can be seen by light scattering). In a third step we were looking deeper into the secondary conformational changes using FT-IR spectroscopy, a common tool used for monitoring α-helix to β-sheet transitions and to disentangle between different β-sheet structures that accompany protein aggregation processes [27,33,41], and which can also be used for pressure-dependent spectroscopic studies. Figure 4 shows FT-IR spectra of various IAPP samples: freshly dissolved IAPP and IAPP aggregated for 3 days at ambient pressure and at 3.5 kbar, respectively (all spectra were taken at room temperature, 25 o C).…”

“…The temperature in the cell was controlled through an external water-circuit. FT-IR spectroscopy has proven to be a powerful technique to determine the secondary structure elements [17,[27][28][29]. The amide I' band (between 1600 and 1700 cm -1 ) was recorded, which is mainly associated with the carbonyl stretching vibration of the amide groups and which is directly related to the backbone conformation and hydrogen bonding pattern of the protein [29].…”