T4 lysozyme

Hayward, S., Berendsen, H.J.C. Systematic analysis of domain motions in proteins from conformational change New results on citrate synthase and T4 lysozyme. Proteins 30 (1998) 144-154. 

Simulation of Small Ligands Bound in T4-lysozyme L99A... 

Extraction of Bound Xenon from Mutant T4-Lysozyme... 

Fig. 2. Work performed to extract xenon from T4 lysozyme L99A in 27 independent simulations of 100 ps each.

Hermans, J., Wang, L. Inclusion of loss of translational and rotational freedom in theoretical estimates of free energies of binding. Application to a complex of benzene and mutant T4-lysozyme. J. Am. Chem. Soc. 119 (1997) 2707-2714... 

Mann, G., Prins, J., Hermans, J. Energetics of forced extraction of ligand Simulation studies of Xe in mutant T4 lysozyme as a simple test system. Bioohys. J., in preparation (1998)... 

Morton, A., Baase, W. A., Matthews, B. W. Energetic origins of specificity of ligand binding in an interior nonpolar cavity of T4 lysozyme. Biochemistry 34 (1995) 8564-8575. 

The critical factor for any method involving an approximation or an extrapolation is its range of application. Liu et al. [15] demonstrated that the approach performed well for mutations involving the creation or deletion of single atoms. The method has also been successfully applied to the prediction of the relative binding affinities of benzene, toluene and o-, p-, and m-xylene to a mutant of T4-lysozyme [16]. In both cases, however, the perturbation to the system was small. To investigate range over which the extrapolation may... 

C Lee. Testing homology modeling on mutant proteins Pi edictmg stiaictural and thermodynamic effects m the Ala98 Val mutants of T4 lysozyme. Folding Des 1 1-12, 1995. 

FIR Faber, BW Matthews. A mutant T4 lysozyme displays five different crystal conformations. Nature 348 263-266, 1990. 

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. 

Figure 17.4 Melting temperatures, Tm, of engineered single-, double-, and tripledisulfide-containing mutants of T4 lysozyme relative to wild-type lysozyme. The red bars show the differences in Tm values of the oxidized and reduced forms of the mutant lysozymes. The green bars for the multiple-bridged proteins correspond to the sum of the differences in Tm values for the constituent single-bridged lysozymes. (Adapted from M. Matsumura et al.. Nature 342 291-293, 1989.)...

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... 

T4 lysozyme has two such cavities in the hydrophobic core of its a helical domain. From a careful analysis of the side chains that form the walls of the cavities and from building models of different possible mutations, it was found that the best mutations to make would be Leu 133-Phe for one cavity and Ala 129-Val for the other. These specific mutants were chosen because the new side chains were hydrophobic and large enough to fill the cavities without making too close contacts with surrounding atoms. 

Karpusas, M., et al. Hydrophobic packing in T4 lysozyme probed by cavity-filling mutants. Proc. Natl. Acad. Sci. USA 86 8237-8241, 1989. 

Anderson DE, Hurley JH, Nicholson H, Baase WA, Mathews BW Hydro-phobic core repacking and aromatic-aromatic interaction in the thermostable mutant of T4 lysozyme Ser 117-Phe. Protein Sci 1993 2 1285-1290. 

Zhang, X.J., and Matthews, B. W. (1994). Conservation of solvent-binding sites in 10 crystal forms of t4 lysozyme. Protein Sd. 3,1031-1039. 

Exit of the virus from the cell occurs as a result of cell lysis. The phage codes for a lytic enzyme, the T4 lysozyme, which causes an attack on the peptidoglycan of the host cell. The burst size of the virus (the average number of phage particies per cell) depends upon how rapidly lysis occurs. If lysis occurs early, then a smaller burst size occurs, whereas slower lysis leads to a higher burst size. The wild type phage exhibits the phenomenon of lysis inhibition, and therefore has a large burst size, but rapid lysis mutants, in which lysis occurs early, show smaller burst sizes. 

Dang, L. X. Merz, K. M. Kollman, P. A., Free-energy calculations on protein stability - Thr1B7-Val157 Mutation of T4 lysozyme, J. Am. Chem. Soc. 1989, 111, 8505-8508. 

Lower and coworkers [199] have investigated the adsorption of T4 lysozyme on colloidal silica. It was observed that the enzymatic activity decreased upon adsorption due to the differences in adsorbed enzyme structure and orientation as well as the electrostatic effects. 

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