Lysozyme folding

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. 

Segel, D. J., Bachmann, A., Hofrichter, J., Hodgson, K. O., Doniach, S., and Kiefhaber, T. (1999). Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering. J. Mol. Biol. 288, 489—499. 

Kiefhaber T (1995) Kinetic traps in lysozyme folding. Proc Natl Acad Sci USA 92 9029-9033 Kim JL, Nikolov DB, Burley SK (1993a) Co-crystal structure of TBP recognizing the minor groove of a TATA elemenL Nature 365 520-527... 

Figure 5.5 Time course for lysozyme folding from the first MS-based millisecond HX experiment. This ability of MS to simultaneously monitor multiple species was critical to building a model for lysozyme folding that involved multiple Intermediates and parallel pathways. Reproduced from Ref [17] with permission American Association for the Advancement of Science...

Bieri, O., Kiefhaber, T. (2001) Origin of apparent fast and non-exponential kinetics of lysozyme folding measured in pulsed hydrogen exchange experiments. Journal of Molecular Biology, 310 (4), 919-935. 

A. Matagne, S. E. Radford, and C. M. Dobson, East and slow tracks in lysozyme folding Insight into the role of domains in the folding process. J. Mol. Biol. 267, 1068-1074 (1997). 

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. 

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 17.3 The polypeptide chain of lysozyme fiom hacteiiophage T4 folds into two domains. The N-terminal domain is of the a + P type, built up from two a helices (red) and a four-stranded antiparallel P sheet (green). The C-terminal domain comprises seven short a helices (brown and blue) in a rather irregular arrangement. (The last half of this domain is colored blue for clarity.)...

No sequence homologies can be detected. This is, perhaps, not surprising. The X-ray structure analysis of lysozyme by Phillips has shown that the polypeptide chain is folded in a way which puts none of the amino acids in sequential vicinity of the catalytic Asp-52 and Glu-37 that are near to the bound substrate. Comparable folding patterns can probably be realized with widely differing arrangements of amino acids, and thus the apparent lack of homologies. 

The particle has a head, within which the viral DNA is folded, and a long, fairly complex tail, at the end of which is a series of tail fibers. During the attachment process, the vims particles first attach to the cells by means of the tail fibers. These tail fibers then contract, and the core of the tail makes contact with the cell envelope of the bacterium. The action of a lysozyme-like enzyme results in the formation of a small hole. The tail sheath contracts and the DNA of the vims passes into the cell through a hole in the tip of the tail, the majority of the coat protein remaining outside. The DNA of T4 has a total length of about 50 /xm, whereas the dimensions of the head of the T4 particle are 0.095 Am by 0.065 fim. This means that the DNA must be highly folded and packed very tightly within the head. 

The hardness of only one type of protein crystal has been found in the literature. It is for lysozyme. This is an enzyme found in egg whites and tears. It destroys bacterial membranes. It is relatively small for a protein molecule, consisting of a chain of 129 amino acids folded into a globule with the volume = 30,000 A3. Its crystals are aggregates of these globular molecules held together by London forces (Stryer, 1988). 

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. 

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