| dc.description.abstract | This thesis seeks to elucidate the role of hydration in the plasticity, stability, and action of lysozyme, and the work reported therein forms part of a long-range programme involving water-mediated transformations, accompanied by the loss of water, when the environmental humidity is systematically reduced. The work done so far as part of this programme is reviewed, and the plan of the work reported in this thesis is outlined in the introductory chapter. The crystallographic and analytical methods used when executing the plan are described in chapter 2.
The crystal structure of a low humidity (88%) form of monoclinic lysozyme with a solvent content as low as 22% by volume has already been determined in this laboratory. However, only an under-refined structure of the native crystal was available. Therefore, this structure was refined using freshly collected data. In the meantime, the refinement of the same structure was reported by another group. The two models differed significantly in the flexible regions of the protein molecule. The two models were reconciled starting from regions of reasonable agreement to produce an improved refined model. The reconciled model was compared with the structure at 88% relative humidity obtained through water-mediated transformation. Parts of the flexible regions of the molecule register significant movements during the transformation. The changes resulting from the transformation from the native to the low humidity forms are pronounced in many of the side chains in the active site region, thus indicating the relationship among hydration, mobility, and enzyme action. The fact that the overall changes in molecular geometry resulting from water-mediated transformation are similar to those which occur during enzyme action further emphasizes this relationship. The details pertaining to the refinement of the native form and its comparative analysis with the low humidity (88%) form and the inhibitor complexes are presented in chapter 3.
Surprisingly, monoclinic lysozyme retains its crystallinity even when the environmental humidity is drastically reduced, thus permitting the structural study of the enzyme at very low levels of hydration. Three very low humidity forms, two of them occurring at a nominal relative humidity of 38% and the other at 5% relative humidity, were characterized. These have unprecedented low solvent contents of 16.9%, 17.6%, and 9.4%, respectively. Admittedly, these forms are likely to contain marginally active or inactive molecules. Yet, it appeared that they might provide useful information on the role of water in the structural integrity and the activity of the enzyme. Therefore, the structures of the very low solvent content forms were determined and refined at moderately high resolution. The relevant structural details pertaining to these forms are presented in chapter 4.
A detailed comparison of the structures of native monoclinic lysozyme (32.3% solvent content) and the forms with solvent contents of 22.2%, 16.9%, 17.6%, and 9.4% forms the subject matter of the final chapter. The reduction in hydration level causes considerable deviations in atomic positions. Understandably, the deviations from the positions in the native structure are the highest in the structure with the lowest level of hydration (5% r.h. form). The deviations are, however, not uniformly distributed in the molecule. They are the highest in the C-terminal segment of domain I and the main loop in domain II, indicating higher flexibility of these regions of the molecule. The extreme flexibility of loops 69-73 and 102-108, and the C-terminal residues (125-129) is particularly striking.
The comparison of water bridges among the structures at different hydration levels clearly indicates that many crucial water-mediated connectivities between different regions of the molecule, which are obviously important for the structural integrity of the protein, are essentially conserved in spite of differences in detail. Attention was focused on the forms with 22.2% and 9.4% solvent contents, as they happen to be structures with the most and the least numbers of located water molecules in the hydration shell. The results clearly demonstrate that the water-mediated tertiary interactions important for the integrity of the protein structure are essentially retained even when the crystal is dehydrated with concentrated sulfuric acid, as in the 5% r.h. form. The very low water content of 9.4% in the 5% r.h. form also perhaps sets the limit beyond which water molecules cannot be removed from the protein molecule without destroying the tertiary structure.
It is known that lysozyme loses its activity when the level of hydration is less than about 0.2 gram per gram of protein. The active site cleft is one of the heavily hydrated regions in all the crystal forms of lysozyme. An earlier study indicated that several water molecules forming part of a network spanning the cleft, remain invariant in five well-refined crystal forms of lysozyme. The number of ordered water molecules in the cleft is, however, much lower in the extremely low solvent content forms than in the forms with a solvent content of 20% or above. Presumably, the water molecules in the cleft are important for enzyme action, admittedly in some as yet unknown way, especially as several of them remain invariant in the normally hydrated forms. The removal of many of these might be a cause for the loss of activity at very low levels of hydration. Another interesting observation pertaining to activity concerns the width of the binding cleft. It was observed that the size of the cleft is much smaller in the 9.4% solvent content form compared to that in all other forms with higher levels of hydration. Thus, the loss of activity accompanying dehydration appears to be caused by the removal of functionally important water molecules from the active site region and the reduction in the size and width of the cleft.
Part of the work presented in the thesis has been reported in the following two publications:
Characterization of lysozyme crystals with unusually low solvent content. Acta Cryst. (1995). D 51, 390-392. (with C. Sudarsanakumar & M. Vijayan)
An X-ray analysis of native monoclinic lysozyme. A case study on the reliability of refined protein structures and a comparison with the low-humidity form in relation to mobility and enzyme action. Acta Cryst. (1996). D 52, 1067-1074. (with C. Sudarsanakumar & M. Vijayan) | |
