Hydration, mobility and action of proteins : an x-ray study involving hen-egg-white lysozyme and human hemoglobin

Abstract

Hydration and mobility of proteins have received considerable attention. It is also recognized that the two are related. A long-range protein crystallographic program in this laboratory is concerned with protein hydration and its consequences and uses primarily an approach involving water-mediated transformations, in which protein crystals undergo reversible transformations with changes in solvent content when the environmental humidity is systematically varied. The work carried out so far has helped in delineating the rigid and flexible regions in the molecules of hen-egg-white lysozyme and bovine ribonuclease A, and in identifying the relatively invariant features in their hydration shells. More importantly, it has resulted in establishing a relation among hydration, mobility, and enzyme action. In the case of lysozyme, it has also been possible to provide a plausible structural explanation for the loss of activity that accompanies extreme dehydration. The work reported in the thesis represents the latest stage in the long-range program. In addition to carrying forward the investigations on lysozyme, the study has been extended to human hemoglobin.

Lysozyme and methemoglobin used in the investigations were purchased, while oxy and deoxyhemoglobin were isolated and purified in the laboratory. Well-known procedures described in the literature were used in most of the crystallization experiments, while crystallization conditions were optimized in a couple of cases. Low-humidity forms of the crystals were produced employing the method used earlier in this laboratory. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU-200 X-ray generator. Data reduction programs XDS and DENZO were used in the work on lysozyme and hemoglobin, respectively. AMoRe was employed for all structure solutions using the molecular replacement method. The structures of lysozyme were refined using X-PLOR, while CNS was used to refine the structures of hemoglobin. In addition to the refinement programs, well-known packages such as FRODO, PROCHECK, MSP, HOMOMGR, and ALIGN were used for model building, structure validation, and analysis of the refined structures.

In order to explore the possible structural changes of lysozyme as a function of pH, the crystal structures of the enzyme were determined at two different pH values, 9.5 and 6.5. Also determined were the crystal structures of the low-humidity (r.h. 88%) variants of the above native forms to compare the effect of the variation of pH with that of variations in hydration. The structure of the orthorhombic form grown at pH 4.5 is already available. Interestingly, the conformation and the mutual orientation of the catalytic residues Glu35 and Asp52 remain unchanged with respect to the change in pH. The changes in molecular geometry and hydration caused by changes in the amount of solvent surrounding protein molecules are more pronounced than those caused by variation in pH. The effect of change in pH on the structure and hydration is also less than that of changes caused by molecular packing. The comparison between the native and low-humidity forms of the orthorhombic pH 9.5 and 6.5 crystals shows that the changes in molecular geometry which accompany the water-mediated transformation to the low-humidity forms are more pronounced in the C-terminal region than in other regions of the molecule. The hydration shell as a whole moves along with the protein molecule during the transformation from native to the low-humidity form, although the locations of only about half the total number of water molecules in the hydration shell remain unchanged.

The reported structures of uncomplexed, unmodified lysozyme, including those determined in this laboratory, account for 20 crystallographically independent copies of the molecule situated in environments involving varying degrees of differences. A detailed comparative study using them leads to delineation of the relatively rigid, moderately flexible, and highly flexible regions of the molecule. Half the binding cleft (Sub-sites D, E, and F) belongs to the rigid region, but the other half (Sub-sites A, B, and C) belongs to a flexible region. There is no marked correlation between relative rigidity and conservation of side-chain conformation, except at the binding site. From the analysis, seven water molecules were identified as invariant. Most of them are involved in important tertiary interactions, while one occurs in the active-site cleft. The study also demonstrates a weak correlation between non-accessibility and rigidity. On average, the level of hydration of polar atoms increases rapidly with accessible atomic surface area but levels off at about 15 Ų at a little over one ordered water molecule per polar protein atom. Of the seven invariant water molecules, one is totally buried within the protein. Only 15 N and O atoms are hydrated in all 20 molecules. Thirteen of these are hydrated by the seven invariant water molecules.

The studies of human hemoglobin encompassed deoxy (T), oxy (R), and met (R) states. However, low-humidity forms of only deoxy and oxy hemoglobin could be studied, as methemoglobin did not survive even a slight reduction in environmental humidity. Contrary to expectations, change in environmental humidity or solvent content of the crystals did not lead to any change in the structure of oxyhemoglobin. On the other hand, surprisingly, the heme geometry in deoxyhemoglobin at low environmental humidity and slightly low solvent content mimics that in oxyhemoglobin. Comparison of the quaternary structures of the two molecules in the crystal asymmetric unit of deoxy low-humidity form with that of T and R states, using the parameters that are widely used to characterize them, revealed that the quaternary association in one of them moves closer to that in the oxygenated molecule, while that of the other moves further away from the oxygenated form towards that in the low-salt deoxy crystals. The difference between the quaternary structures of the two molecules is quite substantial. Hence, it would appear that the molecules in the native low-salt and high-salt deoxyhemoglobin crystals and the two crystallographically independent molecules in the low-humidity form present an ensemble of slightly different quaternary structures representing the T state.

Although low-humidity forms of human methemoglobin could not be prepared, it appeared worthwhile to study the native crystals to explore the similarity of the three crystallographically independent molecules in them with the different reported relaxed (R) states. A comparison of these three molecules with the molecules of the other relaxed states showed that the structures of the three crystallographically independent molecules of human methemoglobin have quaternary structures intermediate between those of the R and R2 structures. The same is true about the disposition of residues in the ‘switch region.’

Thus, it would appear that hemoglobin can access different relaxed states with varying degrees of similarity among them.

The work presented in the thesis has been reported in the following publications:

Sukumar, N., Biswal, B. K. & Vijayan, M. (1999). Structures of orthorhombic lysozyme grown at basic pH and its low-humidity variant. Acta Cryst. D55, 934–937.

Biswal, B. K., Sukumar, N. & Vijayan, M. (1999). Plasticity and hydration of lysozyme: X-ray analysis of a crystal form grown at near-neutral pH and its low-humidity variant and a comparative study of known crystal structures. J. Biosci. 24, Suppl. 1, 36.

Biswal, B. K., Sukumar, N. & Vijayan, M. (2000). Hydration, mobility, and accessibility of lysozyme: structures of a pH 6.5 orthorhombic form and its low-humidity variant and a comparative study involving 20 crystallographically independent molecules. Acta Cryst. D56, 1110–1119.

Biswal, B. K. & Vijayan, M. (2001). Structure of human methemoglobin: the variation of a theme. Curr. Sci. 81, 1100–1105.

Biswal, B. K. & Vijayan, M. (2001). Crystal structures of human oxy and deoxyhemoglobin at different levels of humidity: variability in the T state. Acta Cryst. D, Communicated.