Structural effects of drying and rehydration for enzymes in soils: a kinetics-FTIR analysis of alpha-chymotrypsin adsorbed on montmorillonite.
The effects of desiccation and rehydration cycles encountered by extracellular enzymes in soils are studied on -chymotrypsin adsorbed on montmorillonite. The controlled hygrometric FTIR cell used in this study enables to monitor drying and rehydration steps undergone by the -chymotrypsin-montmorillonite suspension or by the enzyme alone. Relative humidity (RH) determines the amount of deuterated water in the FTIR cell atmosphere. The molar water/protein ratio (W/P) as well as the conformational and solvation states of the enzyme have been determined using H/D exchange monitored by FTIR-transmission spectroscopy. When the W/P ratio decreases from 3500 to approximately 400, unfolding of beta-secondary structure in three different domains involves about 8% of the polypeptide backbone with respect to the most solvated states. Desiccation induces beta-unfolding, which opens channels allowing free vapor water molecules to diffuse into the enzyme at 15% RH. On drying to 0% RH, displacements of internal water (H2O) in the enzyme are demonstrated by reverse peptide isotopic exchanges (COND ==> CONH). Specific beta-structures, only formed in highly solvated states, sequester around 20 internal H2O molecules. Indeed, most of the unfolded secondary structures during the drying step are refolded at W/P approximately 1000 during rehydration. However, self-association hinders the recovery of the initial closed tertiary structure. The pD-dependent structural changes controlling inward and outward water diffusion are suppressed, whether the protein is initially in an adsorbed state or in solution. Changes in secondary structures encountered during desiccation/rehydration cycle are similar for the protein either free or in the adsorbed state. Thus domains that are unfolded by adsorption are not concerned by the desiccation/rehydration cycle.
Noinville
S
S
Laboratoire de Dynamique, Interactions et Réactivité CNRS-Université Paris 6, UMR 7075, 2 rue Henri Dunant, 94320 Thiais, France. silvie.noinville@glvt-cnrs.fr
Revault
M
M
Quiquampoix
H
H
Baron
M H
MH
D000327:Adsorption; D001546:Bentonite / Q000737:chemistry; D002918:Chymotrypsin / Q000737:chemistry*; D007700:Kinetics; D012987:Soil; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water / Q000737:chemistry
D012987:Soil; D014867:Water; D001546:Bentonite; D002918:Chymotrypsin; C117608:alpha-chymotrypsin