Protein crystals, water protons

The Copper Site. In a crystal form of ECAO shown to contain catalytically-active protein (Parsons et al., 1995), the eopper is penta-coordinated in approximate square pyramidal eonfiguration by four basal (equatorial) ligands (His 524, His 526, His 689 and a water [We]) and an apical (axial) water (Wa). The presence of equatorial and axial waters had been first reported by Barker et al. (1979) from EPR, water proton relaxation and kinetic studies on pig plasma amine oxidase and the prediction of histidines and waters as the copper ligands came from EXAFS studies by Scott and Dooley (1985). The equatorial water (We) is labile and not always present. In the HP AO structure (Li et al., 1998) it is present in some, but not all, of the six independent subunits in the same erystal. A comprehensive discussion of the spectroscopic properties of the copper site in amine oxidases, including the exchange rates for the equatorial and axial waters, is given in the review by Knowles and Dooley (1994). 

NMR. Magnetic resonance experiments at low temperatures have been limited largely to proton and deuteron NMR of the water molecules in water-polymer preparations. This is reasonable because of the sensitivity attainable and because the most rapidly moving molecular species (water) is the most easily detected. I will discuss only systems without macroscopic order (e.g., frozen solutions of globular proteins) but the interested reader will find intriguing reports of nmr measurements on protein crystals and on fibrous or layered materials (15,16,17). 

Nonexponential NMR relaxation was reported several years ago for water protons in protein crystals (12). This report has been largely ignored apparently because of a less than adequate explanation that leaned heavily on a chemical exchange model. 

N.m.r. relaxation times have been measured for water protons in cross-linked hen egg-white lysozyme crystals below the freezing point as a function of the mole fraction of protons in the water phase. Data were analysed using a cross relaxation model that eliminated the necessity of postulating long residence times for water molecules in the domain of the protein. 

Finally, we note that all transfers to alcohol-water mixtures or additions of alcohol to crystal mother liquor involve changes in the proton activity of the solution. Care must be taken to ensure that the pH does not change too much, or the crystal may be disrupted. Worse still, the enzymatic activity may be abolished. Control of proton activity in mixed solvents is discussed in Section III,D. If dielectric effects are controlled and pH is properly adjusted, the microenvironment of a crystalline protein will correspond closely to that of aqueous solution at room temperature. Such correspondence is essential for temporal resolution of individual steps in a catalytic reaction. 

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