Three-dimensional structures lysozyme

Both types of mutations have been made in T4 lysozyme. The chosen mutations were Gly 77-Ala, which caused an increase in Tm of 1 °C, and Ala 82-Pro, which increased Tm by 2 °C. The three-dimensional structures of these mutant enzymes were also determined the Ala 82-Pro mutant had a structure essentially identical to the wild type except for the side chain of residue 82 this strongly indicates that the effect on Tm of Ala 82-Pro is indeed due to entropy changes. Such effects are expected to be additive, so even though each mutation makes only a small contribution to increased stability, the combined effect of a number of such mutations should significantly increase a protein s stability. 

Lysozyme is a 14.4-kDa cationic protein (pi > 10) with the ability to kill a wide range of Gram-positive bacteria. It is present in both azurophilic and specific granules of neutrophils and is also found in the granules of monocytes and macrophages, in blood plasma, tears, saliva and airway secretions. In human neutrophils it is present at 1.5-3 jug/106 cells. Since its discovery in 1922 by Fleming, it has been widely studied by protein biochemists, and its three-dimensional structure has been precisely defined. It exerts its ef-... 

The Identities of the sites are shown In Figure 3 and thelrHoca-tlons In the three-dimensional structure of lysozyme can be seen In Figures 4 and 5. 

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. 

Its role is to cut a hole in the bacterial cell wall to permit injection of the virus own DNA. Egg white lysozyme, the first enzyme for which a complete three-dimensional structure was determined by X-ray diffraction,55 is a 129-residue protein. 

The tertiary structure of native proteins is stabilized through hydrophobic interactions in the interior of the three-dimensional structure. The strongly hydrophobic conditions of RPC are known to unfold this conformation. With some species, e.g., with lysozyme, the unfolding is reversible. Here, even RPC does not produce permanently deactivated species but, in general, unfolding causes denaturation. 

Tello, and R. J. Poljak, J. Biol. Chem., 266, 12915 (1991). Crystallographic Refinement of the Three-Dimensional Structure of the FabD 1.3-Lysozyme Complex at 2.5 A Resolution. 

Figure 2. (a) The amino acid sequence of the enzyme lysozyme (from egg white). Blocks enclosing two cysteines (Cys) denote intramolecular covalent cross-links (disulfide bonds). This molecule in crystalline form has the three-dimensional structure sketched in part (b). Note the helical subdomains and sheet substructures formed by nearby extended segments. Reprinted by permission from C. C. F. Blake, Structure of Hen Egg-White Lysozyme, Nature vol. 206 p. 757. Copyright (c) 1967 Macmillan Magazines Ltd. 

The bird lysozymes c, of which chicken egg white lysozyme (CL) is the most extensively studied example, provide an ideal system to recreate evolutionary intermediates and to study structure-function relationships of reconstructed ancestral proteins. Three major considerations qualify the avian lysozyme system for reconstruction of evolutionary pathways (1) the biochemistry of the enzymes has been extensively studied and well characterized,3,4 (2) there are many natural variants available from other birds, and homologous comparisons can be ensured since lysozymes for all game birds are encoded by a single gene,5 and (3) the three-dimensional structure of CL has been resolved at the atomic level, which allows for structural interpretation of the mutational impact.2,4 Proteins representing the ancestral, evolutionarily intermediate, and derived states of chicken and related bird lysozymes are made and characterized as described below. 

A. Models for the Three-Dimensional Structure of a-Lactalbumin (Based on Sequence Homology xvith Lysozyme)... 

Because of the high level of identity in amino acid sequence between lysozyme and a-lactalbumin (see Fig. 10), it was inevitable that interest turned to the three-dimensional structure of a-lactalbumin when the structure of lysozyme was determined in 1965 by the group at the Royal Institution. However, there were unforeseen difficulties in the direct experimental determination, as discussed below. Hence, attention was directed to models for the a-lactalbumin structure based on the coordinates for lysozyme and on energy minimization programs. 

The homology of a-lactalbumin with lysozyme, the similarity in three-dimensional structure and molecular size, etc., are for the well-known c-type (chick) lysozyme. However, there are other forms of lysozyme that catalyze the same reaction as c type. These include insect lysozymes, which are essentially of two types the c type, (Joll s et al., 1979b Eng-... 

In Section VII,B and Fig. 10 we compared the sequences of 13 a-lactalbumins (if the bovine A variant, equine B and C variants, and ovine variant are included), 23 mammalian c-type lysozymes (if donkey, mouse M, bovine stomach 1 and 3, caprine 1 and 2, ovine 1-3, camel 1, deer 2, echidna II, and porcine 1 and 2 are included), and 13 avian c-type lysozymes (if KDIII and PD2 and PD3 are included). Analysis of the sequence differences indicates that, with the recent considerable increase in the number of lysozymes sequenced, there has been an appreciable decrease in the numbers of residues that are invariant in lysozymes as well as for both proteins. Nevertheless, there is still significant overall homology (—35%) between a-lactalbumin and c-type lysozyme. From the similarities in amino acid sequences, three-dimensional structures, intron—exon patterns, etc., there can be little doubt that the concept of divergence is still valid for these proteins. What is controversial are the rate of evolution and the details of the way in which ct-lactalbumin arose, although it is conceded generally that the mechanism involves gene duplication. 

A tremendous amount has been achieved by the application of X-ray crystallography in determining the three-dimensional structure of a-lactalbumin and lysozyme, and in the case of lysozyme, the mode of its catalytic action. Nevertheless, despite the tremendous advances, there are still areas of this mechanism that are not fully understood. This is especially true of the comparative mode of action of c-, g-, and phage-type lysozymes. 

T. O. Fischmann, G.A. Bentley, T.N. Bhat, G. Boulot, R.A. Mariuzza, S.E. Phillips, D. Tello, and R.J. Poljak. 1991. Crystallographic refinement of the three-dimensional structure of the FabDl.3-lysozyme complex at 2.5-A resolution J. Biol. Chem. 266 12915-12920. (PubMedl... 

The first crystal structure of an enzyme, that of lysozyme, was determined by David C. Phillips and coworkers " in 1965. The most striking feature in the three-dimensional structure of lysozyme is a prominent cleft that traverses one face of the molecule. The X-ray structure of lysozyme complexed with a three-residue oligosaccharide showed that this cleft was, indeed, the substrate-binding site. The crystal structure of this complex provided the first three-dimensional model for how enyzmes work. 

This volume has been written for those chemists and biochemists who may never themselves do X-ray diffraction analyses of crystals, but who need to be able to understand the results of such studies on structures of immediate interest to them. The fields of structural biology and chemistry have blossomed in the years since X-ray diffraction was discovered in 1912. For example, the three-dimensional structures of benzene, graphite, the alkali halides, the boron hydrides, the rare gas halides, penicillin, vitamin Bjj, hemoglobin, lysozyme, transfer RNA, and the common-cold rhinovirus have been determined and, in each case, the results have greatly increased our understanding of fundamental chemistry and biochemistry. 

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