Abstract
Temporal binding of urea to lysozyme was examined using X-ray diffraction of single crystals of urea/lysozyme complexes prepared by soaking native lysozyme crystals in solutions containing 9 M urea. Four different soak times of 2, 4, 7 and 10 hours were used. The five crystal structures (including the native lysozyme), refined to 1.6 Å resolution, reveal that as the soaking time increased, more and more first-shell water molecules are replaced by urea. The number of hydrogen bonds between urea and the protein is similar to that between protein and water molecules replaced by urea. However, the number of van der Waals contacts to protein from urea is almost double that between the protein and the replaced water. The hydrogen bonding and van der Waals interactions are initially greater with the backbone and later with side chains of charged residues. Urea altered the water-water hydrogen bond network both by replacing water solvating hydrophobic residues and by shortening the first-shell intra-water hydrogen bonds by 0.2 Å. These interaction data suggest that urea uses both ‘direct’ and ‘indirect’ mechanisms to unfold lysozyme. Specific structural changes constitute the first steps in lysozyme unfolding by urea.
Highlights
Space group Unit cell (Å) Total reflections Unique reflections Multiplicity Completeness (%) Mean I/sigma(I) Wilson B-factor (Å2) R-merge (%) R-meas (%) CC1/2 CC* R-work R-free Number of non-hydrogen atoms Macromolecules Ligands Water Protein residues RMS bonds (Å) RMS angles (°) Ramachandran favoured (%) Ramachandran outliers (%) Clash score Average B-factor (Å2) Macromolecules Ligands Solvent
The average B-factors for the protein, the urea molecules and the water molecules are significantly higher in 10 h compared to others (Table 1), consistent with a possible onset of denaturation in 10 h
This result is in contrast to constancy of protein-solvent hydrogen bonds found in Molecular Dynamics (MD) simulations on barnase[41]
Summary
Space group Unit cell (Å) Total reflections Unique reflections Multiplicity Completeness (%) Mean I/sigma(I) Wilson B-factor (Å2) R-merge (%) R-meas (%) CC1/2 CC* R-work R-free Number of non-hydrogen atoms Macromolecules Ligands Water Protein residues RMS bonds (Å) RMS angles (°) Ramachandran favoured (%) Ramachandran outliers (%) Clash score Average B-factor (Å2) Macromolecules Ligands Solvent. High-resolution crystal structures that map urea binding to lysozyme as a function of time. These results should help in evaluating MD simulation data on protein unfolding by urea[11,41]. The complex structures reveal good electron density for a total of forty-seven urea molecules occupying twenty-one different positions replacing forty-nine water molecules in the first solvent shell. The urea molecules form far more van der Waals interactions with the protein compared to displaced water molecules, in agreement with results of MD simulations[35]. Urea-binding has led to the loss of a few intra-protein hydrogen bonds, and this, we propose, represents the start of the pathway of lysozyme denaturation by urea
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