Protein crystallography water molecules

The input to a minimisation program consists of a set of initial coordinates for the system. The initial coordinates may come from a variety of sources. They may be obtained from an experimental technique, such as X-ray crystallography or NMR. In other cases a theoretical method is employed, such as a conformational search algorithm. A combination of experimenfal and theoretical approaches may also be used. For example, to study the behaviour of a protein in water one may take an X-ray structure of the protein and immerse it in a solvent bath, where the coordinates of the solvent molecules have been obtained from a Monte Carlo or molecular dynamics simulation. 

On the other hand, as biological molecules become larger their tendency to be associated with water molecules, metal ions, and other materials increases. Crystalline proteins, for example, routinely contain 27-65 % of the solvent used for their crystallisation 183). Such associated materials may be difficult to locate by crystallography and it may become a question of terminology whether such molecules should be regarded as inclusion complexes, non-specific aggregates, or merely contaminated biomolecules. 

Water is present in protein cavities as individual molecules, water chains, and clusters. Indeed, tightly bound waters can be resolved in X-ray crystallography experiments. Water molecules in larger cavities, especially those with a hydrophobic surface, are mobile and less readily resolved. In some proteins, such as the cytochrome b(f complex or cytochrome c oxidase, bound water molecules tend to form water chains. These water molecules provide hydrogen-bonded relays for proton transfer, and they may mediate donor-to-acceptor electronic coupling (2-6). 

The structure of this water-selective membrane pore protein (Fig. 3a and 3b) represents the highest resolution stmcture obtained from electron crystallography to date (53). Data were obtained for Aquaporin-O in double-layered 2-D crystals, and its staggering 1.9-A resolution clearly reveals water molecules within the pore. The data also reveal associated lipids, allowing key protein-lipid interactions to be modeled. 

Figure 1 In a QM/MM calculation, a small region is treated by a quantum mechanical (QM) electronic structure method, and the surroundings treated by simpler, empirical, molecular mechanics. In treating an enzyme-catalysed reaction, the QM region includes the reactive groups, with the bulk of the protein and solvent environment included by molecular mechanics. Here, the approximate transition state for the Claisen rearrangement of chorismate to prephenate (catalysed by the enzyme chorismate mutase) is shown. This was calculated at the RHF(6-31G(d)-CHARMM QM-MM level. The QM region here (the substrate only) is shown by thick tubes, with some important active site residues (treated by MM) also shown. The whole model was based on a 25 A sphere around the active site, and contained 4211 protein atoms, 24 atoms of the substrate and 947 water molecules (including 144 water molecules observed by X-ray crystallography), a total of 7076 atoms. The results showed specific transition state stabilization by the enzyme. Comparison with the same reaction in solution showed that transition state stabilization is important in catalysis by chorismate mutase78.

Figure 27 A 2Fobs-Foa 0 electron density map of Aspergillus flavus urate oxidase contoured at the 1 a- level (M. Spano, unpublished). The absolute value of the electron density is actually not useful in macromolecular crystallography what matters are the relative values. Electron density values are commonly given as multiples of the standard deviation ct, and where there are electrons, there are atoms. The protein atoms are shown in sticks and the red spheres represent ordered water molecules.

This model was established by Chen and Schoenbom when identifying water molecules and ions in a crystal of CO myoglobin protein by combining neutrons and x-ray crystallography. The surface structure exhibits 85 water molecules and 5 ions while the access path of the CO to the heme is devoid of bound water. Thus there is a subtle interplay between the hydrophilic and hydrophobic phenomena that control the stability of the folded state. 

Interactions between specific solvent molecules and protein atoms are important for protein structure and function. Water molecules participate directly in many enzymatic reactions, including those catalyzed by the large class of enzymes that hydrolyze peptide bonds. X-ray crystallography has identified waters that appear to be an integral part of the structure of a protein. Some of... 

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