Reversible and irreversible denaturation processes in globular proteins: from collective to molecular spectroscopic analysis

Abstract

Different spectroscopic techniques were applied for studying the structural properties of lysozyme in salt-free aqueous solutions. The results of vibrational and Brillouin scattering measurements were compared to obtain both single-molecule and collective properties of the solutions. The characterization of the protein system, from the conformation of the polypeptide chain to the exposure of side chains to the solvent and the arrangement of the solution network, was then achieved in the range 25-85 • C. Through the analysis of the indole breathing mode, a different environment for the six tryptophan residues of an unfolded lysozyme could be evidenced. Short and long exposures to high temperatures were used to modulate the competition between the thermally induced reversible and irreversible denaturation processes. These different thermal treatments were applied to distinguish between the effects of global unfolding of the single molecule from those of self-aggregation and gel formation. It has been observed that clusterization occurs at melting temperatures with slow kinetics; also, aggregates evolve from the completely unfolded state of the protein and lead to a sensitive increase in viscosity. This effect probably hinders any further conformational rearrangement of the molecules in the aggregate; thus as a consequence, the disordered structure of clusters does not change to give the β-sheet organization, characteristic of filaments or fibrils.

Amyloid Self-Assembly of Lysozyme in Self-Crowded Conditions: The Formation of a Protein Oligomer Hydrogel

“…The sample is similar to the opaque particulate gel formed by LYS after incubation at 65 °C and pH = 12 when the low protein charge favors the rapid formation of amorphous aggregates . We remark that the FTIR AI signal is not sensitive to aggregates of an amorphous nature . The initial loosening of β-sheet contacts starts at lower temperatures (10 °C or more) than previously observed for analogous structures formed in different environments (presence of DMSO or ethanol and higher pH), , suggesting that the thermal stability of amyloid oligomers depends on the solvating conditions.…”

“…34 We remark that the FTIR AI signal is not sensitive to aggregates of an amorphous nature. 77 The initial loosening of β-sheet contacts starts at lower temperatures (10 °C or more) than previously observed for analogous structures formed in different environments (presence of DMSO or ethanol and higher pH), 72,73 suggesting that the thermal stability of amyloid oligomers depends on the solvating conditions. Likely, the high surface charge of lysozyme at pH = 1.8 75 might induce the formation of relatively small oligomers with a lower thermal stability.…”

Relaxation dynamics of a protein solution investigated by dielectric spectroscopy

“…[21] the peak was ascribed to the temperature dependence of hydrophobic interactions within the protein molecules. Also reversible denaturation effects may be considered [75].…”

Recent advances in linear and nonlinear Raman spectroscopy. Part VII

“…Sassi and co‐workers carried out studies of reversible and irreversible denaturation processes in globular proteins using a variety of spectroscopic probes. The results of vibrational and Brillouin scattering measurements were compared to obtain both single‐molecule and collective properties of the solutions . Schaefer, Fox, and Harris used confocal Raman microscopy to monitor the membrane polymerization and thermochromism of individual, optically trapped diacetylenic phospholipid vesicles.…”