Disulfide lysozyme

Figure 6.6 Schematic diagram of the structure of the enzyme lysozyme which folds into two domains. One domain is essentially a-helical whereas the second domain comprises a three stranded antiparallel p sheet and two a helices. There are three disulfide bonds (green), two in the a-helical domain and one in the second domain.

Lysozyme from bacteriophage T4 is a 164 amino acid polypeptide chain that folds into two domains (Figure 17.3) There are no disulfide bridges the two cysteine residues in the amino acid sequence, Cys 54 and Cys 97, are far apart in the folded structure. The stability of both the wild-type and mutant proteins is expressed as the melting temperature, Tm, which is the temperature at which 50% of the enzyme is inactivated during reversible beat denat-uration. For the wild-type T4 lysozyme the Tm is 41.9 °C. 

The ROA spectra of partially unfolded denatured hen lysozyme and bovine ribonuclease A, prepared by reducing all the disulfide bonds and keeping the sample at low pH, together with the ROA spectra of the corresponding native proteins, are displayed in Figure 5. As pointed out in Section II,B, the short time scale of the Raman scattering event means that the ROA spectrum of a disordered system is a superposition of snapshot ROA spectra from all the distinct conformations present at equilibrium. Because of the reduced ROA intensities and large... 

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. 

Other groups within the protein may affect excited states. Disulfide bonds quench the excited states of tryptophan. For instance, at 77 K the phosphorescence lifetime of native lysozyme is low, 1.4s reduction of the disulfide bonds or denaturation gave the typical phosphorescence lifetime of 5.6 s.(49) Therefore, the absence of phosphorescence at room temperature from this protein is likely to be due to quenching of both the singlet and the triplet state. 

The extent of formation of protein disulfides with time was determined by withdrawing aliquots which were acidified to pH 5.5 and alkylated with N-ethylmaleimide. The disulfide content of the peptide was determined after its isolation. Formation of two intrapeptide disulfide bonds proceeded at the same rate (within experimental error) as formation of the first two disulfides in reduced lysozyme. The first-order rate constant for these two processes (0.5 min-1) was eight times that describing the rate of oxidation of reduced lysozyme in the presence of 6 M guanidinium chloride, suggesting substantial specificity in the process in absence of denaturant. An additional indication of specificity was the finding that 13-105 reached its maximum of two —S—S— bonds in less than 20 minutes, retaining one reduced thiol from 20 to 240 minutes. For subsequent studies this material was S-alkylated with N-ethylmaleimide. 

The kinetics of disulfide formation, the demonstration of specific binding, and the immunochemical results all support the conclusion that native-like structure results from the oxidative folding of reduced peptide 13-105. These three independent lines of evidence support the conclusion that lysozyme has a continuous chain independent assembly region somewhere in the sequence 13-105. 

Now that about 70 different disulfides have been seen in proteins and more than 20 of those have been refined at high resolution, it is possible to examine disulfide conformation in more detail, as it occurs in proteins. Many examples resemble the left-handed small-molecule structures extremely closely Fig. 46 shows the Cys-30-Cys-115 disulfide from egg white lysozyme. The x > Xs and x dihedral angles and the Ca-Ca distance can be almost exactly superimposed on Fig. 45 the only major difference is in Xi All of the small-molecule structures have Xi close to 60°. Figure 47 shows the Xi values for halfcystines found in proteins. The preferred value is -60° (which puts S-y trans to the peptide carbonyl), while 60° is quite rare since it produces unfavorable bumps between S-y and the main chain except with a few specific combinations of x value and backbone conformation. 

Fig. 46. A left-handed spiral disulfide from hen egg white lysozyme, viewed from a direction similar to Fig. 45.

One ionic bond that often helps establish tertiary structure is a disulfide bond between two cysteine side chain groups—for instance, in the enzyme lysozyme as shown in Figure 2.10. Lysozyme is not a metalloprotein, such as will be studied in this text, but it is a small enzyme and is illustrative of some secondary and tertiary structures found in the more complex molecules described in the following chapters. Lysozyme protects biological species from... 

A spacer was used to link captopril via a disulfide bond to the LMWP lysozyme. Conjugation of captopril to lysoz5me resulted in a 6-fold increase in captopril accumulation in the rat kidney (Eigure 5.9b) [77]. This modest enrichment, as compared to that achieved with naproxen-lysozyme, was due to fact that, in contrast to naproxen, free captopril is cleared very efficiently by the kidney itself. Thus, delivery via lysozyme reabsorption only leads to a limited improvement of renal accumulation of captopril. 

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. 

In small proteins, hydrophobic residues are less likely to be sheltered in a hydrophobic interior—simple geometry dictates that the smaller the protein, the lower the ratio of volume to surface area. Small proteins also have fewer potential weak interactions available to stabilize them. This explains why many smaller proteins such as those in Figure 4—18 are stabilized by a number of covalent bonds. Lysozyme and ribonuclease, for example, have disulfide linkages, and the heme group in cytochrome c is covalently linked to the protein on two sides, providing significant stabilization of the entire protein structure. 

Lysozyme is a natural antibacterial agent found in tears and egg whites. The hen egg white lysozyme (Mr 14,296) is a monomer with 129 amino acid residues. This was the first enzyme to have its three-dimensional structure determined, by David Phillips and colleagues in 1965. The structure revealed four stabilizing disulfide bonds and a cleft containing the active site (Fig. 6-24a see also Fig. 4-18). More than five decades of lysozyme investigations have provided a detailed picture of the structure and activity of the enzyme, and an interesting story of how biochemical science progresses. 

Three tyrosines react with cyanogen fluoride in the neutral range (Gobrinoff 1967). While all four residues react with the tetranitromethane, only two are nitrated (Habeeb and Atassi 1971 Denton and Ebner 1971). These observations are generally consistent with the proposed model (Warme et al 1974). The disulfide bonds in a-lactal-bumin, as predicted from the expanded model, are more rapidly reduced and, therefore, more accessible than in lysozyme (Iyer and Klee... 

Figure 25-15 Lysozyme from hen egg-white showing the amino-acid sequence (primary structure) and the four intrachain disulfide bridges. [Adapted from D. C. Phillips, Sc/. Amer. 5, 215 (1966).]...

The structure of wool is more complicated than that of silk fibroin (Figure 25-13) because wool, like insulin (Figure 25-8) and lysozyme (Figure 25-15), contains a considerable quantity of cystine, which provides —S—S— (disulfide) cross-links between the peptide chains. These disulfide linkages play... 

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