title: Influence of urea and guanidine hydrochloride on lysozyme stability and thermal denaturation; a correlation between activity, protein dynamics and conformational changes.
abstract: The effect of urea and guanidine hydrochloride (GuHCl) on lysozyme stability has been investigated using activity measurements, microcalorimetry and Raman spectroscopy in the low-frequency and amide I regions. Raman investigations on lysozyme dissolved in H(2)O and D(2)O in the presence of up to 10 M denaturants have revealed direct binding between the protein and both denaturants. The analysis of isotopic exchanges in the amide I region allows the identification of binding sites as hydrophilic and hydrophobic groups, respectively, for urea and GuHCl. The weak loss of activity of lysozyme in the presence of urea (∼15% maximum) is mainly assigned to a transformation of the tertiary structure corresponding to a molten globule state without unfolding of α-helix structures, in contrast to GuHCl which clearly induces conformational changes, associated with a larger loss of activity (40% maximum). The denaturing power of urea and guanidine hydrochloride on lysozyme has been related to the solvent and protein dynamics, reflecting direct interaction between denaturants and protein. It clearly appears that solvent dynamics control protein dynamics, and the significant hardening of the dynamics of GuHCl aqueous solutions is considered responsible for its important denaturing power. The comparison between the low-frequency spectra of solvents and lysozyme aqueous solutions in the absence and presence of different types of additives (urea, GuHCl, trehalose) reveals the Raman signature of the hydration water dynamics. This comparison points out the exclusion of trehalose around the protein surface.
authors.affiliation: Univ Lille Nord de France, F-59000 Lille, France. alain.hedoux@univ-lille1.fr
authors.forename: Alain
authors.initials: A
authors.lastname: Hédoux
authors.forename: Stefanie
authors.initials: S
authors.lastname: Krenzlin
authors.forename: Laurent
authors.initials: L
authors.lastname: Paccou
authors.forename: Yannick
authors.initials: Y
authors.lastname: Guinet
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Flament
authors.forename: Juergen
authors.initials: J
authors.lastname: Siepmann
mesh_terms: D002152:Calorimetry, Differential Scanning; D019791:Guanidine / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D013059:Spectrum Analysis, Raman; D013696:Temperature; D014508:Urea / Q000737:chemistry*
chemical_list: D014508:Urea; D009113:Muramidase; D019791:Guanidine