Human lysozyme

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.)...

Figure 3 Anharmonicity factor versus quasi-harmomc mode number from a 200 ps vacuum simulation of BPTI. It can be seen that beyond about the 200th mode the anharmonicity factors are about 1.0, indicating harmomcity. Those below mode number 200 show progressively greater anharmonicity factors, indicating that they span a space within which energy barriers are crossed. A similar picture was found for a I ns simulation of human lysozyme m water [61]. (Adapted from Ref. II.)...

One of the main attractions of normal mode analysis is that the results are easily visualized. One can sort the modes in tenns of their contributions to the total MSF and concentrate on only those with the largest contributions. Each individual mode can be visualized as a collective motion that is certainly easier to interpret than the welter of information generated by a molecular dynamics trajectory. Figure 4 shows the first two normal modes of human lysozyme analyzed for their dynamic domains and hinge axes, showing how clean the results can sometimes be. However, recent analytical tools for molecular dynamics trajectories, such as the principal component analysis or essential dynamics method [25,62-64], promise also to provide equally clean, and perhaps more realistic, visualizations. That said, molecular dynamics is also limited in that many of the functional motions in biological molecules occur in time scales well beyond what is currently possible to simulate. 

Figure 4 DynDom [67] analysis of the first two normal modes of human lysozyme. Dark grey and white indicate the two dynamic domains, separated by the black hinge bending region. The vertical line represents a hinge axis that produces a closure motion in the first normal mode. The horizontal line represents a hinge axis that produces a twisting motion in the second normal mode. (Adapted from Ref. 68.) The DynDom program is available from the Internet at http //md. chem.rug.nl/ steve/dyndom.html.

The ROA spectra of native and prehbrillar amyloidogenic human lysozyme are displayed in Figure 7, together with a MOLSCRIPT diagram of the native structure. The ROA spectrum of the native protein is very similar to that of hen lysozyme (Fig. 5). However, large changes have occurred in the ROA spectrum of the prehbrillar intermediate. In particular, the positive 1340 cm-1 ROA band assigned to hydrated... 

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. 

A recent NMR study of the structure and dynamics of two amyloido-genic variants of human lysozyme (Chamberlain et al., 2001) showed that, although one variant destabilized the /6-domain much more than the other, it had no greater propensity to form amyloid fibrils. It was concluded that the increased ability of the variants to access substantially unfolded conformations of the protein is the origin of their amy-loidogenicity. This appears to reinforce the conclusions from ROA that a destabilized a-domain is involved in fibril formation. 

The conformational plasticity supported by mobile regions within native proteins, partially denatured protein states such as molten globules, and natively unfolded proteins underlies many of the conformational (protein misfolding) diseases (Carrell and Lomas, 1997 Dobson et al., 2001). Many of these diseases involve amyloid fibril formation, as in amyloidosis from mutant human lysozymes, neurodegenerative diseases such as Parkinson s and Alzheimer s due to the hbrillogenic propensities of a -synuclein and tau, and the prion encephalopathies such as scrapie, BSE, and new variant Creutzfeldt-Jacob disease (CJD) where amyloid fibril formation is triggered by exposure to the amyloid form of the prion protein. In addition, aggregation of serine protease inhibitors such as a j-antitrypsin is responsible for diseases such as emphysema and cirrhosis. 

It has been suggested recently that PPII helix may be the killer conformation in such diseases (Blanch et al., 2000). This was prompted by the observation, described in Section III,B, of a positive band at 1318 cm-1, not present in the ROA spectrum of the native state, that dominates the ROA spectrum of a destabilized intermediate of human lysozyme (produced by heating to 57°C at pH 2.0) that forms prior to amyloid fibril formation. Elimination of water molecules between extended polypeptide chains with fully hydrated 0=0 and N—H groups to form... 

As indicated in Table 2.1, most of the promoters used in plant tissue culture have been based on the constitutive cauliflower mosaic virus (CaMV) 35S promoter. In contrast, inducible promoters have the advantage of allowing foreign proteins to be expressed at a time that is most conducive to protein accumulation and stability. Although a considerable number of inducible promoters has been developed and used in plant culture applications, e.g. [32-37], the only one to be applied thus far for the production of biopharmaceutical proteins is the rice a-amylase promoter. This promoter controls the production of an a-amylase isozyme that is one of the most abundant proteins secreted from cultured rice cells after sucrose starvation. The rice a-amylase promoter has been used for expression of hGM-CSF [10], aranti-trypsin [12, 29, 38, 39] and human lysozyme [30]. 

C.-G. Golander, V. Hlady, K. Caldwell, and J. D. Andrade, Absorption of human lysozyme and adsorbate enzyme activity as quantified by means of total internal reflection fluorescence, 125I labeling and ESCA, Colloids Surf. 50, 113-130 (1990). 

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. 

A preliminary description of the sequence of the amino acids in human lysozyme has been published, and it may be compared with the sequence in hen egg-white lysozyme.116 The human enzyme, which is 2.7 to 3.0 times as active as the hen s enzyme, was obtained... 

Barel, A. 0., Prieels, J. P., Maes, E., Looze, Y. and Leonis, J. 1972. Comparative physicochemical studies of human a-lactalbumin and human lysozyme. Biochim. Biophys. Acta 257, 288-296. 

The active site is in a cleft between a large domain with a nonpolar core and a smaller (3-sheet domain that contains many hydrogen-bonded polar side chains (Figs. 12-3,12-4). Human lysozyme has a similar structure and properties.57-59 The T4 lysozyme has an additional C-terminal domain whose function may be to bind the crosslinking peptide of the E. coli peptidoglycan. Goose lysozyme is similar in part to both hen lysozyme and T4 lysozyme. All three enzymes, as well as that of our own tears, may have evolved from a common ancestral protein.60 On the other hand, Streptomyces erythaeus has developed its own lysozyme with a completely different structure.61 An extensive series of T4 lysozyme mutants have been studied in efforts to understand protein folding and stability.61-63... 

Fig. 7.6. Figure 7.6. Backscattered ICP Raman (IR f IL) ancj j oA (IR - IL) spectra of (a) human lysozyme in the native state, (b) human lysozyme in the low pH molten globule state, and (c) the T-A-l peptide from wheat glutenin. Adapted from references 45 and 46...

Figure 9.3. Restriction map produced by Webcutter. The partial restriction map shows the nucleotide sequence of human lysozyme gene submitted to Webcutter using options for all restriction endonucleases with recognition sites equal to or greater than six nucleotides long and cutting the sequence 2 6 times (at least 2 times and at most 6 times). The restriction profile (map) is returned if Map of restriction sites is selected for display. The tables by enzyme name and by base pair number can be also returned if displays for Table of sites, sorted alphabetically by enzyme name Table of sites, sorted sequentially by base pair number are chosen.

Figure 10.2. Removal of noncoding repetitive elements with RepeatMasker. The nucleotide sequence encoding human lysozyme is submitted to RepeatMasker and the sequence with masked(X) noncoding repetitive elements is returned (partial sequence is shown here).

Figure 10,6. Prediction of exons with FEX at Sanger Centre. The nucleotide sequence of DNA encoding human lysozyme (5648 bp) is submitted to Sanger Centre for exon prediction with FEX. The output shows number of predicted exons, locations, and amino acid sequences of translates.

Perform search button. The request returns the positions and scores (threshold) of the donor/acceptor sites (e.g., DNA encoding for human lysozyme with 5648 bp) ... 

Figure 10.8. Gene identification by Procrustes. The nucleotide sequence encoding human lysozyme is used as a query sequence to identify its gene structure against known protein sequence (i.e., pig lysozyme protein). The output includes sequence aignment of the source (predicted translate) versus target protein (pig lysozyme).

Figure 12.5. Summary page of PDBsum. The summary page for human lysozyme complex (1LZC) shows hyperlinks to various structure analyses.

Figure 15.7. Mage desktop window. The desktop window of MAGE program consists of three component windows (graphic window, caption window, and text window) as shown for human lysozyme (1 Iza.pdb, 11zb.pdb, and 11zc.pdb). The displayed structural features can be turned on and off via checkboxes. The check marks (

M3. Maron, E., and Bonavida, B., A sensitive immunoassay for human lysozyme in biological fluids. Biochim. Biophys. Acta 229, 273-275 (1971). 

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