Neutron diffraction protein hydration

Protein crystals contain between 25 and 65 vol% water, which is essential for the crystallisation of these biopolymers. A typical value for the water content of protein crystals is 45% according to Matthews et al. l49,150). For this reason it is possible to study the arrangement of water molecules in the hydration-shell by protein-water and water-water interactions near the protein surface, if one can solve the structure of the crystal by X-ray or neutron diffraction to a sufficiently high resolution151 -153). 

The best and most direct methods to observe the hydration of a macromolecule are X-ray and neutron diffraction analyses carried out at high resolution to better than 1.8 A. In the crystals of proteins, the macromolecules are heavily hydrated so that between 20Vo and 909o of the total volume are solvent. In fact, despite their well-defined crystal morphology, crystalline proteins more resemble concentrated protein solutions than the solid state. 

X-ray and neutron diffraction measurements on polyion hydration give the number of water molecules involved per repeat group in the structures. About one water molecule per repeat group is the result for polymethyl methacrylate. The results of hydration for a variety of proteins are given in Table 2.31. 

C-Phycocyanin is abundant in blue-green algae. Nearly 99% deuterated samples of this phycobiliprotein were isolated from the cyanobacteria that were grown in perdeuterated cultures [46] (99% pure D2O) at Argonne National Laboratory. This process yielded deuterated C-phycocyanin proteins (d-CPC) that had virtually all of the H—C bonds replaced by H—C bonds. One can obtain a lyophilized sample that is similar to amorphous solids as determined by neutron diffraction [43]. As it has been defined in previous papers [47-49], the level of hydration h = 0.5 corresponds to 100% hydration of C-phycocyanin, which leads to a coverage of about 1.5 monolayers of water molecules on the surface of the protein [50]. 

The protein-solvent interface was studied in an explicit solvent environment of 3182 water molecules by MD simulations performed on metmyoglobin [31].Both the structure and dynamics of the hydrated surface of myoglobin are similar to that obtained by experimental methods calculating three-dimensional density distributions, temperature factors and occupancy weights of the solvent molecules. On the basis of trajectories they identified multiple solvation layers around the protein surface including more than 500 hydration sites. Properties of theoretically calculated hydration clusters were compared to that obtained from neutron and X-ray data. This study indicates that the simulation unified the hydration picture provided by X-ray and neutron diffraction experiments. 

Dobson, C. M. (2001) The structural basis of protein folding ans its links with human disease, Philos. Trans. R. Soc. Lond. B356, 133-145 Chen, X., and Schoenbom, B. P. (1990) Hydration in protein crystals. A neutron diffraction analysis of carbonmonoxymyoglobin, Acta Crystallographica B46, 195-208... 

Perkins (2001) discussed x-ray and neutron diffraction from the hydration shell of proteins, but did not refer to the experimental results of Svergun et al. (1998). Instead he used the estimate of 0.0245 nm for a water molecule in the hydration shell by Gerstein and Chothia (1996) to explain the apparent different partial molar volume measured densitomefrically for proteins in solution with the values calculated from their amino acid contents. 

Fairly recently, the water (D20)-protein correlations at the surface of a fully deuterated amorphous protein C-phycocyanin have been studied by neutron diffraction as functions of temperature and hydration level [53]. 

Recent progress in X-ray diffraction of protein crystals in the diamond anvil cell will also make it possible to obtain quantitative information on the cavities [42, 43]. Optical spectroscopy [44] and neutron scattering [45] should also be valuable tools to probe the role of cavities. High-pressure molecular dynamics simulations should also allow estimating the contributions of the hydration and the cavities. High-pressure simulations on the small protein, bovine pancreatic trypsin inhibitor, indicate an increased insertion of water into the protein interior before unfolding starts to occur [46,47]. 

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