Lysozyme molecules

Figure 2 Internal RMSF of residues (average over heavy atoms) determined for human lysozyme by the X-ray normal mode refinement method applied to real X-ray data (heavy curve), m comparison with results from a normal mode analysis on a single isolated lysozyme molecule (lightweight curve). (From Ref. 33.)...

Recently Blake et al.153) made such studies in the case of human (HL) and tortoise egg-white (TEWL) lysozyme based on crystallographic refinements at 1,5 and 1,6 A resolution, respectively. By these investigations they attempted to obtain information on the perturbations of water structure in the hydration shell by neighboured protein molecules and by high salt concentrations as well as on the degree of order of the bound water. The authors came to the conclusion that the number of ordered water molecules are 128 in TEWL and 140 in HL, whereas the overall content is made up of 650 and 350 water molecules per lysozyme molecule. 

FIGURE 1119 The lysozyme molecule is a typical enzyme molecule. Lysozyme is present in a number of places in the body, including tears and the mucus in the nose. One of its functions is to attack the cell walls of bacteria and destroy them. This "ribbon" representation shows only the general arrangement of the atoms to emphasize the overall shape of the molecule the ribbon actually consists of amino acids linked together (Section 19.13). 

The effect of pH on the protein adsorption on CMK-3 was also investigated [152], The monolayer adsorption capacities obtained under various pH conditions are plotted in Figure 4.12, where the maximum adsorption was observed in the pH region near the isoelectric point of lysozyme (pi of about 11). Near the isoelectric point, the net charges of the lysozyme molecule are minimized and would form the most compact assembly. A similar pH effect was also seen in the adsorption of cytochrome c on CM K-3. Although the nature of the surface of mesoporous silica and... 

In an earlier experiment, Jori et al. (14) reported that methionyl residues are important in maintaining the tertiary structure of lysozyme. The introduction of a polar center into the aliphatic side chain of methionine, as a consequence of the conversion of the thioether function to the sulfoxide, may bring about a structural change of the lysozyme molecule which, in turn, reduces the catalytic efficiency. When ozonized lysozyme was treated with 2-mercaptoethanol in an aqueous solution according to the procedure of Jori e al. (14), the enzyme did not show any increase in its activity. This may be explained in two ways. In one, such reactions are complicated by many side reactions, e.g. sulfhydryl-disulfide interchange, aggregation and precipitation of the modified enzyme (24-26). In the other, the failure to recover the activity of the enzyme may by associated with the extensive oxidation of other residues. 

Fig. 2. —Atomic Arrangement in the Lysozyme Molecule in the Neighborhood of the Cleft with a Hexa-N-acetylchitohexaose Molecule Shown Bound to the Enzyme. (The main polypeptide chain is shown speckled, and NH and CO are indicated by line and full shading, respectively. Sugar residues A, B, and C are as observed in the binding of tri-N-acetylchitotriose. Residues D, E, and F occupy positions inferred from model building. It is suggested that the linkage hydrolyzed by the action of the enzyme is between residues D and E.) [Reprinted from Brookhaven Symp. Biol., 21, 120 (1968).]...

Competitive Inhibition Enzyme Assays. Estimates of antibody-lysozyme dissociation constants can be obtained by taking advantage of the fact that most monoclonal antibodies efficiently inhibit enzymatic activity.3 5 The combining site of HyHEL-10, which is presented as an example, also has been demonstrated by X-ray crystallography to overlap a portion of the catalytic site of lysozyme.7 A constant concentration of lysozyme is incubated with varying amounts of antibody, and amounts of free (unbound) lysozyme molecules are estimated by the proportion of catalytic activity remaining. The assay assumes that the addition of Micrococcus lysodeikticus cell walls and concurrent dilution of the antibody-antigen mixture do not disturb the equilibrium. 

V-acetylmethionine in quantities equal to LZM. Inactivation of LZM also may be restricted upon addition of /V-acetylcystine or /V-acetyltryptophan. The /V-acetylo-tryptophan competes with LZM for the oxidizing agent. Amino compounds binding hypochlorite to relatively stable chloramines, such as leucine, lysine, and taurine, do not protect LZM from MPO-mediated reaction. Oxidation of tryptophan residues in lysozyme molecules treated with HOC1 occurs along with a decrease of lysozyme spectral properties at 280 nm. 

Another possibility for explaining second-order kinetics is that each lysozyme molecule, being trapped in the cellular debris, is, in effect, eliminated from the reaction mixture after it participates in a single lysis. The lysozyme so involved then behaves kinetically as if it were a reactant rather than a catalyst, since it cannot separate from the product in order to participate in another reaction. However, the extent to which lysozyme behaves in this way, as a second reagent, to account for second-order kinetics, remains to be assessed. 

In summary, the main structural features of the domestic hen egg-white lysozyme molecule (see Fig. 3) are ... 

The antigenic determinants of the native lysozyme molecule appear to include most, if not all, of the surface residues, as evidenced by numerous studies (reviewed critically by Benjamin et al., 1984). This point of view contrasts with that of Atassi and Lee (1978), who claimed a limited antigenicity, based on a study of surface-simulated peptides. The sites delineated by the latter workers do not include Arg-68 (Fainanu et al., 1974), the loop region in general, or any of several other segmental regions previously demonstrated to function in this capacity, such as residues 1—20 and 123-129 (see Benjamin et al., 1984). 

In addition to the influence of the detergent on crystallization, the introduction of additional protein components has proven to be successful. In several cases, an antibody fragment was cocrystallized with the membrane protein to provide essential crystal contacts for crystallization (55-57). The protein itself can be engineered with an insertion to provide crystal contacts as demonstrated by the recent success of human 32 adrenergic G-protein-coupled receptor stmcture, in which a lysozyme molecule was engineered into one of the loops (58). 

The lysozyme molecule was determined to have three antigenic sites with residues coming from widely separated portions of the polypeptide chain the residues proposed as contacting and those synthesized to produce a linear sequence considered as best simulating the active site are seen in Fig. 15. In some instances the peptide synthesized in the reverse direction —for example, using the C-terminal amino acid of the hypothesized determinant as the amino terminus—was used as a control. In some instances, the sequence was considered to have directionality whereas in others it did not. 

E-fS ES ES. The standard deviation of the distribution, (Atopen ) = 8.3 2ms, reflects the distribution bandwidth. For the individual T4 lysozyme molecules examined under the same enz unatic reaction conditions, we found that the first and second moments of the single-molecule topen distributions are homogeneous, within the error bars. The hinge-bending motion allows sufficient structural flexibility for the enzyme to optimize its domain conformation the donor fluorescence essentially reaches the same intensity in each turnover, reflecting the domain conformation reoccurrence. The distribution with a defined first moment and second moment shows typical oscillatory conformational motions. The nonequilibrium conformational motions in forming the active enzymatic reaction intermediate states intrinsically define a recurrence of the essentially similar potential surface for the enzymatic reaction to occur, which represents a memory effect in the enzymatic reaction conformational dynamics [12,41,42]. 

To characterize the binding and the motions of the placed single T4 lysozyme molecules on cell walls, we used single-molecule fluorescence polarization measurements. The orientation of the single-molecule transition dipole can be probed by either linear polarized excitation or linear polarized emission. In this work, the excitation light was unpolarized. The emission was split into orthogonal (s polarized /i and p polarized I2) polarizations and detected by two photon detectors [22]. The intensity trajectories probed at the two orthogonal polarizations are shown in Fig. 24.9c (upper panel). The polarization P is defined as ... 

FIGURE 13.13. Diagram of the human lysozyme molecule, with residues having displacements greater than 0.2 M outlined by parallel lines. The active site cleft runs almost vertically down the center of the drawing. (Courtesy D. C. Phillips)... 

By using a surface radioactivity technique, the penetration of the hydrophobic and flexible 1-14C-acetyl--casein and the rigid and globular 1-14C-acetyl-lysozyme molecules into phospholipid monolayers in different physical states was monitored. The adsorption of ff-casein to lecithin mono-layers is described by a model in which it is assumed that the protein condenses the lecithin molecules so that the degree of penetration is a function of the lateral compressibility of the phospholipid monolayer. The interaction of ff-casein with phospholipid monolayers is dominated by the hydrophobicity of the macromolecule, but lysozyme tends to accumulate mostly beneath phospholipid monolayers in this situation, electrostatic interactions between the lipid and protein are important. 

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