Lysozyme variants

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. 

The caprine and ovine stomach lysozyme variants differ from bovine C2 as follows ... 

Boodi DR, et al. Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 1997 385 787-793. 

Shioi S, Imoto T, Ueda T (2004). Analysis of the early stage of the folding process of reduced lysozyme using all lysozyme variants containing a pair of cysteines. Biochem. 43 5488-5493. 

D.R. Booth, M. Sunde, V. Bellotti, C.V. Robinson, W.L. Hutchinson, RE. Frazer, P.N. Hawkins, C.M. Dobson, S.E. Radford, C.C.F. Blake, and M.B. Pepys, Instability, Unfolding and Aggregation of Human Lysozyme Variants Underlying Amyloid Fibrillogenesis. Nature, 385, 787-793,1997. 

Bower, C.K., Xu, Q., and McGuire, J. (1998). Activity losses among T4 lysozyme variants after adsorption to colloidal silica. Biotechnol. Bioeng. 58 (6), 658-662. 

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. 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

Double-quantum coherence (DQC) was used to measure interspin distances in eight doubly labelled derivatives of T4 lysozyme.25 Distances between 20 and 47 A with distance distributions of 1.0 to 2.8 A were measured. For some variants two conformations were detected with distances that differed by 5 to 5.4 A. It was shown that a few measurements of large distances provided significant constraints on protein structure. 

Sources of Naturally Occurring Evolutionary Variant Lysozymes... 

Determination of Effects of Amino Acid Replacements among Evolutionary Variant Lysozymes on Antibody Affinity... 

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. 

The CL variants are constructed by site-directed mutagenesis of the cDNA for chicken egg white lysozyme and heterologously expressed in yeast behind a regulated hybrid promoter.6... 

Carbonic anhydrase, a-lactalbumin, trypsin inhibitor, oval-bumn, conalbumin, hemoglobin variants Ribonuclease, insulin, a-lactalbumin Lysozyme, a-chymotrypsinogen, ribonuclease A, cytochrome c... 

The propensities of folded proteins to aggregate will therefore depend on the accessibility of such aggregation-prone species, a conclusion that is clearly demonstrated by detailed studies of the amyloidogenic mutational variants of lysozyme, which we have found to decrease the stability and cooperativity of the native state (Fig. 13.4) [40-43]. Indeed, these experiments show that the effect of the disease-associated mutations is to decrease the energy difference between the native state and the intermediates populated in the normal folding of the protein, such that the latter are accessible to a much greater extent in the variants than in the wild-type protein [40]. The large mass of evidence now accumulated from studies of lysozyme has provided detailed... 

We will see in Sections VII and X that the recent elucidation by X-ray crystallography of the binding sites for Ca(II) in baboon a-lactalbumin has led to a flurry of studies of potential binding by variants of lysozyme in a wide range of species. 

All a-lactalbumins and c-type lysozymes have eight half-cystine residues (four disulfide bridges). There have been no reports of the presence of cysteine. There has been one report of a bovine a-lactalbumin having six half-cystines (Barman, 1973). As far as we know, no further work has been done on this variant. 

Two variants of a-lactalbumin, caprine and ovine, have no Met residues, indicating that this residue plays no direct role in the lactose synthase system. Of the lysozymes only baboon milk and pigeon egg-white lysozymes have no Met residues. 

Two other variants of pig stomach mucosa lysozyme—namely, I and 2—have been isolated and sequenced by Jolles et al. (1989). 

A second variant, echidna milk lysozyme II, has been identified by Hopper and McKenzie (1974) and later sequenced (see Section VII). 

Jolles et al. (1984) determined the 129-residue sequence of one of the three variants (variant 2) of the three c-type lysozymes isolated by Dobson et al. (1984) from bovine stomach mucosa. The sequence is given here. However, residue 98 has been altered from the original designa-don of Lys to His in accordance with the revision by R Jolles and J. Jolles (quoted by Prager and Wilson, 1988). Note (6) Work by Jolles et al. (1990) on the other bovine variants and two variants of caprine stomach mucosa lysozyme, and by Irwin and Wilson (1990) on three ovine variants, was in press at the time of writing and the following information was made available courtesy of Ellen Prager and Allan Wilson. 

The 129-residue sequence of axis deer stomach mucosa lysozyme was determined by Jollds et al. (1989). There now appear to be two variants. The sequence given in Fig. 10 is that of variant 1. Irwin and Wilson (1990) have concluded that variant 2 differs from variant 1 as follows ... 

The sequences of echidna lysozyme I from the milk of Tachyglossus aculeatus multiacuJeatus and echidna lysozyme II from the milk of Tachyglossus aculeatus aculeatus have been determined by Teahan et al. (1990 see also Teahan, 1986). Note (8) The sequences for variants I and II differ as follows ... 

---