Crystals lysozyme

Very recently, Sadler et al. have prepared a half-sandwich arene ruthenium(ll) derivative of crystallized lysozyme by slow diffusion of the complex [(ri -p-cymene)-RuCl2(H20)] in tetragonal lysozyme crystals (80). High resolution X-ray crystallography of the modified crystals unexpectedly revealed that a single arene ruthenium entity happened to be coordinated to the enzyme by interaction with the imidazole ring borne by the H1S15 residue. This is the first example of this kind of protein structure reported in the literature. 

FIR Faber, BW Matthews. A mutant T4 lysozyme displays five different crystal conformations. Nature 348 263-266, 1990. 

The structure of lysozyme in the complex is the same as that in crystals of free lysozyme, and no conformational changes are seen even in the regions... 

Fremont, D.H., Monnaie, D., Nelson, C.A., Hendrickson, W.A., Unanue, E.R. Crystal structure of I-A in complex with a dominant epitope of lysozyme. Immunity 8 305-317, 1998. 

Radian, E.A., et al. 5tructure of an antibody-antigen complex. Crystal structure of the HyHEL-10 Fab-lysozyme complex. Proc. Natl. Acad. Sci. USA 86 5938-5942,... 

Since there are so few direct packing interactions between protein molecules in a crystal, small changes in, for example, the pH of the solution can cause the molecules to pack in different ways to produce different crystal forms. The structures of some protein molecules such as lysozyme and myoglobin have been determined in different crystal forms and found to be essentially similar, except for a few side chains involved in packing interactions. Because they are so few, these interactions between protein molecules in a crystal do not change the overall structure of the protein. However,... 

At best, van der Waals interactions are weak and individually contribute 0.4 to 4.0 kj/mol of stabilization energy. ITowever, the sum of many such interactions within a macromolecule or between macromolecules can be substantial. For example, model studies of heats of sublimation show that each methylene group in a crystalline hydrocarbon accounts for 8 k[, and each C—IT group in a benzene crystal contributes 7 k[ of van der Waals energy per mole. Calculations indicate that the attractive van der Waals energy between the enzyme lysozyme and a sugar substrate that it binds is about 60 k[/mol. 

From crystal-structure analysis of hen-egg lysozyme and of its complex with the competitive inhibitor tri-Af-acetylchitotriose, the following conclusions were drawn the active site consists of a cleft containing six sub-sites, A to F, of which each could accommodate a) -( 1 — 4)-linked A-acetylglucosa-... 

Crystal-structure analysis of Taka amylase A gave similar results, in that it showed that it had an extended cleft which could accommodate six, or possibly seven, a-( 1 — 4)-linked glucose units and two oppositely placed acidic amino acids (Asp-206 and Glu-230) which could interact with the bound substrate similarly to Asp-52 and Glu-35 in lysozyme. 

Fig. 5. Backscattered Raman and ROA spectra of native (top pair) and reduced (second pair) hen lysozyme, and of native (third pair) and reduced (bottom pair) bovine ri-bonuclease A, together with MOLSCRIPT diagrams of the crystal structures (PDB codes llse and lrbx) showing the native disulfide links. The native proteins were in acetate buffer at pH 5.4 and the reduced proteins in citrate buffer at pH 2.4. The spectra were recorded at 20°C. 

Fig. 7. Backscattered Raman and ROA spectra of native human lysozyme in acetate buffer at pH 5.4 measured at 20°C (top pair), and of the prehbrillar intermediate in glycine buffer at pH 2.0 measured at 57°C (bottom pair), together with a MOLSCRIPT diagram of the crystal structure (PDB code ljsf) showing the tryptophans. 

Zhang, X.J., and Matthews, B. W. (1994). Conservation of solvent-binding sites in 10 crystal forms of t4 lysozyme. Protein Sd. 3,1031-1039. 

The hardness of only one type of protein crystal has been found in the literature. It is for lysozyme. This is an enzyme found in egg whites and tears. It destroys bacterial membranes. It is relatively small for a protein molecule, consisting of a chain of 129 amino acids folded into a globule with the volume = 30,000 A3. Its crystals are aggregates of these globular molecules held together by London forces (Stryer, 1988). 

A shear modulus of about 1 GPa has been measured for wet lysozyme. Thus its Chin-Gilman parameter is about 0.02 which is large compared with metals and small compared with covalent crystals. 

K. Koizumi, M. Tachibana, H. Kawamoto, and K. Kojima, Temperature Dependence of Microhardness of Tetragonal Hen-egg-white Lysozyme Single Crystals, Phil. Mag., 84,2961 (2004). 

Fields, B.A., F.A. Goldbaum, W. Dall Acqua, E.L. Malchiodi, A. Cauerhff, F.P. Schwarz, X. Ysem, R.J. Poljak, and R.A. Mariuzza. 1996. Hydrogen bonding and solvent structure in an antigen-antibody interface. Crystal structures and thermodynamic characterization of three Fv mutants complexed with lysozyme. Biochemistry 35 15494-15503. 

Since use of increasing amounts of cosolvents as antifreeze could perturb the conformation and thus the activity of lysozyme, a number of experiments were carried out to try to determine conditions for investigating lysozyme reactions and lysozyme-substrate intermediates in cooled mixed solvents as a preliminary to similar investigation by X-ray diffraction on crystals. Work began with an estimate of the solubility of... 

These results suggest that the crystallographic determination of the structure of a productive enzyme-substrate complex is feasible for lysozyme and oligosaccharide substrates. They also provide the information of pH, temperature, and solvent effects on activity which are necessary to choose the best conditions for crystal structure work. The system of choice for human lysozyme is mixed aqueous-organic solvents at -25°C, pH 4.7. Data gathered on the dielectric constant, viscosity, and pH behavior of mixed solvents (Douzou, 1974) enable these conditions to be achieved with precision. 

FIGURE 6.22 (a) The white-light image of lysozyme crystals, (h) The retrieved Raman spectrum of a crystalline lysozyme (top) and the retrieved Raman spectrum of lysozyme in solution (50 mg/mL hottom). 

Schwartz, A. M., and Berglund, K. 2000. In situ monitoring and control of lysozyme concentration during crystallization in a hanging drop. J. Cryst. Growth 219 753-60. 

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