Evidence of a two-stage thermal denaturation process in lysozyme: A Raman scattering and differential scanning calorimetry investigation

Abstract

Raman spectroscopy (in the low-frequency range and the amide I band region) and modulated differential scanning calorimetry investigations have been used to analyze temperature-induced structural changes in lysozyme dissolved in 1H2O and 2H2O in the thermal denaturation process. Low-frequency Raman data reveal a change in tertiary structure without concomitant unfolding of the secondary structure. Calorimetric data show that this structural change is responsible for the configurational entropy change associated with the strong-to-fragile liquid transition and correspond to about 1/3 of the native-denaturated transition enthalpy. This is the first stage of the thermal denaturation which is a precursor of the secondary structure change and is determined to be strongly dependent on the stability of the hydrogen-bond network in water. Low-frequency Raman spectroscopy provides information on the flexibility of the tertiary structure (in the native state and the transient folding state) in relation to the fragility of the mixture. The unfolding of the secondary structure appears as a consequence of the change in the tertiary structure and independent of the solvent. Protein conformational stability is directly dependent on the stability of the native tertiary structure. The structural transformation of tertiary structure can be detected through the enhanced 1H/2H exchange inhibited in native proteins. Taking into account similar features reported in the literature observed for different proteins it can be considered that the two-stage transformation observed in lysozyme dissolved in water is a general mechanism for the thermal denaturation of proteins.

Raman spectroscopic and low-temperature calorimetric investigation of the low-energy vibrational dynamics of hen egg-white lysozyme

“…It is worth to emphasize that, a linear frequency trend of the coupling coefficient for biological systems has long been supposed in analogy to glassy systems [22,9] but, to our knowledge, this is the first time that this assumption has been experimentally confirmed.…”

“…1, clearly, the Raman signal appears to be characterized by a strong quasi-elastic contribution at frequency below 20 cm -1 and by the presence of two bands in the frequency range between 20<ν<300 cm -1 , where harmonic vibrations of lysozyme are expected to dominate [9]: the first band is really closed to the quasi-elastic tail, while the second one is placed at higher frequencies (behind 50 cm -1 ) and appears as a shoulder on the right side of the first bump. A fitting procedure has been performed by using two Lorentzian shapes and the theoretical curves well reproduce both polarized and depolarized Raman scattering spectra (see fig.…”

Terahertz underdamped vibrational motion governs protein-ligand binding in solution

“…Raman and THz-TDS spectroscopy of crystalline lysozyme and other proteins shows peaks in the 20-100 cm À 1 (B1-3 THz) range 23,[26][27][28][29] . However, these are very sensitive to crystal packing and hydration levels, and may be associated with phonon modes of the crystal lattice, rather than biologically relevant phenomena.…”

“…Although recent THz-TDS experiments on lysozyme crystals have successfully identified underdamped delocalized vibrational modes in the terahertz range 23 , the crystal packing and hydration level modify the protein dynamics, and it remains impossible to show the biochemical relevance of such modes. Inelastic neutron scattering 24,25 and spontaneous Raman scattering [26][27][28][29] have been used to study lysozyme in solution and do not suffer from water absorption. However, these techniques become unreliable at low frequencies (o1 THz) due to the very strong Rayleigh peak from elastic scattering, and again it has not been possible to assign biochemical relevance to any peaks observed.…”

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

“…In a series of interruptionincubation DSC experiments, Luo et al established a method to distinguish between two-state and non-two-state (lysozyme and bovine serum albumin respectively) denaturation processes [30]. A combination of Raman spectroscopy and temperature-modulated scanning calorimetry shows support for a two-stage process during the thermal denaturation of lysozyme [31]. An initial change in the tertiary structure without concurrent unfolding of secondary structure, followed by unfolding of the secondary structure was observed.…”