Lysozyme stability

Binding to lysozyme stabilizes a conformation in site D compatible with the alternative mechanism. (i) Hg of residue D does not interact with residue E but forms a strong hydrogen bond to the mainchain 0 of residue 57, a residue involved with the unusual buried p turn in lysozyme. (ii) The value of 0 between D and E, which was -54 in the initial structure, stabilized at -62 , near the optimum of -60 for stereoelectronic assistance. 

Kang, F., Jiang, G., Hinderliter, A., DeLuca, P. R, and Singh, J. (2002), Lysozyme stability in primary emulsion for PLGA microsphere preparation Effect of recovery methods and stabilizing excipients, Pharm. Res., 19, 629-633. 

Perry, L.J. Wetzel, R. Disulfide bond engineered into T4 lysozyme stabilization of the protein toward thermal inactivation. Science 1984, 226 (4674), 555-557. 

Ghalanbor, Z. Korber, M. Bodmeier, R. Improved lysozyme stability and release properties of poly(lactide-co-glycolide) implants prepared by hot-melt extrusion. Pharm. Res. 2010, 27(2), 371-379. 

Recently, Kollman and coworkers calculated the effect of a methyl substitution on the a versus P carbon atom of the unnatural amino acid (S-2-amino-3-cyclopentylpropanoic acid) in position 133 on T4 lysozyme stability. The authors used AMBER 4.1 and compared the results obtained by thermodynamic integration method with the results obtained by newer methods. 2 5 The study determined that the methyl group substitution on the a carbon stabilizes T4 lysozyme by 1.8 kcal/mol, whereas the methyl substitution on the P carbon destabilizes the protein by 0.4 kcal/mol. The result is in good agreement with the less CPU-intensive method, pictorial representation of free energy changes (PROFEC). 

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. 

We will discuss three different approaches to engineer a more thermostable protein than wild-type T4 lysozyme, namely (1) reducing the difference in entropy between folded and unfolded protein, which in practice means reducing the number of conformations in the unfolded state, (2) stabilizing tbe a helices, and (3) increasing the number of bydropbobic interactions in tbe interior core. 

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. 

Figure 17.5 Diagram of the T4 lysozyme stmcture showing the iocations of two mutations that stabilize the protein stmcture by providing eiectrostatic interactions with the dipoles of a helices. (Adapted from H. Nicholson et al.. Nature 336 651-656, 1988.)...

Mutants that fill cavities in hydrophobic cores do not stabilize T4 lysozyme... 

At best, van der Waals interactions are weak and individually contribute 0.4 to 4.0 kj/mol of stabilization energy. ITowever, the sum of many such interactions within a macromolecule or between macromolecules can be substantial. For example, model studies of heats of sublimation show that each methylene group in a crystalline hydrocarbon accounts for 8 k[, and each C—IT group in a benzene crystal contributes 7 k[ of van der Waals energy per mole. Calculations indicate that the attractive van der Waals energy between the enzyme lysozyme and a sugar substrate that it binds is about 60 k[/mol. 

Both attractive forces and repulsive forces are included in van der Waals interactions. The attractive forces are due primarily to instantaneous dipole-induced dipole interactions that arise because of fluctuations in the electron charge distributions of adjacent nonbonded atoms. Individual van der Waals interactions are weak ones (with stabilization energies of 4.0 to 1.2 kj/mol), but many such interactions occur in a typical protein, and, by sheer force of numbers, they can represent a significant contribution to the stability of a protein. Peter Privalov and George Makhatadze have shown that, for pancreatic ribonuclease A, hen egg white lysozyme, horse heart cytochrome c, and sperm whale myoglobin, van der Waals interactions between tightly packed groups in the interior of the protein are a major contribution to protein stability. 

On the basis of the above, the rate acceleration afforded by lysozyme appears to be due to (a) general acid catalysis by Glu (b) distortion of the sugar ring at the D site, which may stabilize the carbonium ion and the transition state) and (c) electrostatic stabilization of the carbonium ion by nearby Asp. The overall for lysozyme is about 0.5/sec, which is quite slow (Table... 

Theoretical Studies of Enzymic Reactions Dielectric, Electrostatic and Steric Stabilizations of the Carbonium Ion in the Reaction of Lysozyme A. Warshel and M. Levitt Journal of Molecular Biology 103 (1976) 227-249... 

In this paper, we study the stabihty of the carbonium ion intermediate formed in the cleavage of a glycosidic bond by lysozyme. It is found that the electrostatic stabilization is an important factor in increasing the rate of the reaction step that leads to the formation of the carbonium ion intermediate. Steric factors, such as the strain of the substrate on binding to lysozyme, do not seem to contribute significantly. 

GENERAL ACID CATALYSIS AND ELECTROSTATIC STABILIZATION IN THE CATALYTIC REACTION OF LYSOZYME... 

FIGURE 6.6. The type of model compounds that were used to estimate the electrostatic stabilization in lysozyme (the only hydrogen atom shown, is the one bonded to the oxygen). Such molecules do not show a large rate acceleration due to electrostatic stabilization of the positively charged carbonium transition state. However, the reaction occurs in solution and not in a protein-active site, and the dielectric effect is expected to be very different in the two cases. 

Warshel A, Levitt M (1976) Theoretic studies of enzymic reactions dielectric electrostatic and steric stabilization if the carboniumion in the reaction of lysozyme. J Mol Bio 103 227... 

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