Crystal structures lysozyme

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,... 

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. 

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. 

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. 

Lysozyme is an enzyme that hydrolyzes some bacterial cell-walls, the bacterium used for the assay being Micrococcus lysodeikticus. Lysozyme is found in a wide variety of species and locations, including bacteriophages, blood, egg white, gastric secretions, milk, nasal mucus, papaya, sputum, and tears. The outstanding achievement in this field has been the elucidation of the crystal structures of some of the lysozyme-substrate complexes. 

The distance measured by EPR is the point dipole distance between the two paramagnetic centres. When applied to structures of spin-labelled biomolecules the desired distance is between sites on the biomolecules. A key question is the conformation and conformational flexibility of the spin label. X-ray crystal structures of four spin-labelled derivatives of T4 lysozyme have been reported.54 Preferred rotational conformations of the linkage between the cysteine introduced by site-directed mutagenesis and the spin label were observed. The electron density associated with the nitroxide ring in different mutants was inversely correlated with the mobility of the label observed in fluid solution EPR spectra. 

Hen egg white lysozyme is a small protein of Mr 14 500 and 129 amino acid residues. This enzyme was introduced in Chapter 1, where it was pointed out that examination of the crystal structure of the enzyme stimulated most of the solution studies. Hen egg white lysozyme has the distinction of being the first enzyme to have had its structure solved by x-ray crystallography.207 It is an atypical member of the hexosaminidase class of glycosyl transfer enzymes. It catalyzes the hydrolysis of substrates with retention of stereochemistry. T4 lysozyme was for many years thought to have the same fold and mechanism of lysozyme, despite there being no sequence homology. But it has now been found that the T4 enzyme has inversion of configuration and so operates by a different mechanism.208,209 A mechanism proposed for the enzymatic reaction was based on the structure of the... 

Mariuzza RA (1996) Crystal structure of the complex of the variable domain of antibody D1.3 and the turkey egg with lysozyme a novel conformational change in antibody CDF-L3 selects for antigen, J Mol Biol, 257 889-894... 

In summary, we have shown that site-directed mutagenesis is an important tool which allows precise investigation into the detailed chemistry of protein-protein interaction. HEL has proved to be ideal for these studies. Monoclonal antibodies, X-ray crystal structures of protein complexes, and evolutionary lysozyme variants found in nature provide an excellent system for understanding the molecular basis of protein recognition. Site-directed mutagenesis offers the ability to alter a protein at a specific site. Interpretation of the results within the context of an X-ray crystal structure allows the quantitative assignment of the free energy contributions from individual amino adds to the stability of the complex. 

Because internal water molecules are in mostly apolar environments, their hydrogen bonds are often strong and well defined. The average O- -O distance is 2.89 0.21 A for the internal water molecules in lysozyme, carboxypeptidase, cytochrome c, actinidin, and penicillopepsin. As with the small molecule crystal structures, the water molecules are involved in three or four hydrogen bonds, with 48% engaged in three and 37% in four interactions. 

Internal water molecules tend to form clusters. In general, internal water molecules in protein structures are not found isolated but are assembled in clusters. Their hydrogen-bonding scheme could be derived in actinidin (Fig. 19.13), in lysozyme, and in penicillopepsin, based oh the assumption that water molecules act as double donors and acceptors. In some of the protein structures, which have been analyzed in greater detail, an internal water is associated with three acceptor sites indicating three-center bonding as observed in the amino acid zwitterion crystal structures (see Part IB, Chap. 8). 

About 300 water molecules are sufficient to cover the lysozyme surface. This is a remarkably small amount of water. Calculations based on the crystal structure of lysozyme show that the surface is about 6000 A in area (Lee and Richards, 1971 Shrake and Rupley, 1973). Thus, each water covers, on average, about 20 A, which is twice the projection of a water molecule packed as in the liquid. Since 20 is the most area a water molecule can cover and maintain hydrogen bonding, there can be no multilayer water. Moreover, whatever arrangements there are at the surface must integrate simply into the bulk water namely, there is no B shell of disordered water, required to interface the water adjacent to the protein surface with the bulk solvent. 

In any event it was the analysis of baboon a-lactalbumin crystals for which the first X-ray crystal structure was produced, initially at 0.6 nm (6 A) and 0.45 nm (4.5 A) (Phillips etal., 1987 Smith etal., 1987). More recently, the structure has been refined at 0.17-nm (1.7-A) resolution, enabling comparison with the high-resolution c-type lysozyme structure (Acharya et al., 1989) (see Fig. 7). 

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