Mutated residues

Thirty percent of the tumor-derived mutations are in L3, which contains the single most frequently mutated residue, Arg 248. Clearly the interaction between DNA and the specific side chain of an arginine residue inside the minor groove is of crucial importance for the proper function of p53. It is an open question whether this interaction is needed for the recognition of specific DNA sequences, or is required for the proper distortion of the DNA structure, or a combination of both. Other residues that are frequently mutated in this region participate in interactions with loop L2 and stabilize the structures of loops L2 and L3. Mutations of these residues presumably destabilize the structure so that efficient DNA binding can no longer take place. 

A more recent study was based on horse heart cytochrome c and Rhodobacter sphaeroides cytochrome c oxidase (18). Here, the Lys55 residue was ruthenated and the cytochrome c oxidase was mutated at several surface sites. A model of the complex between cytochrome c and subunit II of cytochrome c oxidase, the heme c and Cua cofactors, and the mutated residues is shown in Fig. 8. 

Fig. 2. Schematic view of mutation points in cytochrome Pac-551. The position of a carbon atoms of the mutated residues are shown by closed circles. Haem iron is indicated as double lined circle. The atomic co-ordinated for Pac-551 were taken from Protein Data Bank (code 451C). Reprinted with permission from J. Biol. Chem., Vol. 274, J. Hasegawa, H. Shimahara, M. Mizutani, S. Uchiyama, H. Arai, M. Ishii, Y. Kobayashi, S. J. Ferguson, Y. Sambongi and Y. Igarashi, 1999, p. 37,533.

Another significant difference between the large- and small-subunit enzymes lies in the fact that the heme d of HPII and PVC is flipped 180° relative to the heme b moiety of BLC, MLC, SCC-A, and PMC (Fig. 13). This is clearly a function of the residues that form the heme pocket, although attempts to force a change in heme orientation in HPII by mutating residues that interact with the heme were imsuccessful. The heme is situated in the (3-barrel and has interactions with the wrapping domain and with the amino-terminal arm of the R-related subunit. The dimensions of the pocket demand that heme bind in its final conformation and that flipping once inside the pocket not be possible. 

Match obvious catalytic or structural residues, hydrophobic regions, glycine and proline residues. Modify (mutate) residues accordingly. 

Is easy id predict the structure of a point mutant. They also determined a set of simple rules that can be used to predict the structure of a mutated residue given the structure of the wH4 ypc protein. These rules are described in Figure 3. It is to be expected—although it is not yet proven—that this simple scheme can be extrapolated to model building by homology in cases of high homology. 

Figure 3 Decision scheme for the prediction of the conformation of a mutated residue. This scheme is based on two observations (1) A point mutation seldom leads to large alterations in the overall structure of the protein. The mutated residue adapts to the structure of the rest of the protein rather than the other way around. (2) Most residues sit lit the statistically preferred conformation, and when exceptions occur, they can normally be explaimi on the basis of hydrogen-bonding patterns. (Adapted from Ref. 37.)...

Non-additivity arises from the simultaneous disruption of coupled residues by multiple mutations. Mutations affecting some functions, such as protein-protein and DNA-protein interactions, tend to be remarkably additive, while others, such as mutations in the catalytic site, tend to be non-additive (Jencks, 1981 Wells, 1990). Mutational studies on T4 lysozyme (Zhang et al., 1995) and catalase I (Trakulnaleamsai et al., 1995) indicate that thermostability is largely additive, and therefore relatively uncoupled. Non-additivity is most commonly observed when the mutated residues are close in space and large, or when chemically disparate side chains are introduced. 

LiCata and Ackers report that mutations that are not directly in contact can be non-additive (1995). They find that most mutations exhibit some degree of non-additivity that cannot be explained by short-range disruptions. A structural study of mutants of pNB esterase generated by directed evolution supports this observation (Spiller et al., 1999 see chapter by Orencia, Hanson, and Stevens in this volume). In this case, the influence of a mutation was realized through small backbone shifts, spatially distant from the mutated residue. Non-additivity can result when these perturbed regions overlap (Skinner and Terwilliger, 1996). 

Fig. 8. MolScript (Kraulis, 1991) diagram of the three-dimensional structure of />NB esterase variant 8G8 (Spiller et al., 1999). Mutated residues are shown in black ball-and-stick. Catalytic residues are shown in white ball-and stick. Black portions indicate stabilized loop regions.

To increase the enantioselectivity of these myoglobin metalloenzymes, Lu and co-workers have successfully utilized a covalent linkage approach [62], In an earlier attempt a Mn(III)-salen complex was incorporated into apo-myoglobin by mutating residue 103 to cysteine, followed by modification with a methane thiosulfonate derivative of Mn(III)(salen) (Figure 5.16). This catalyst showed sulfoxidation activity however, the ee was only 12 %. As such a low ee might be a result of the ability of the bound ligand to exist in multiple conformations within the protein cavity, it was hypothesized that the rotational freedom of the salen complex could be limited if it was anchored at... 

Figure 6-1. Complex formed between an immunoglobulin and a hen egg lysozyme highlighting the mutated residues with a vdW representation. The null-spots are represented in yellow (relative binding energy <2.0 kcal/mol), the warm-spots in orange (relative binding energy between 2.0 and 4.0 kcal/mol), and the hot spots in red (residues with a relative binding energy higher than 4.0 kcal/mol)...

Question Does the abolition of activity following mutagenesis unequivocally indicate a catalytic role for the mutated residue ... 

Thus a change in a peptide molecular mass indicates the position of the mutation whereas the difference between the native peptide molecular mass and that of the mutant peptide allows determination of the nature of the amino acid that has mutated as long as only one possibility exists. And in some cases, it is even possible to determine the position of the mutation in the peptide when the mutated residue is found only once in the peptide. Otherwise, MS/MS is necessary. 

Fig. 7. Structural model of CCR5 dimer association presented as a ribbon representation. TMl, TM2, and TM4 (labeled al, a2 and o4) participate directly in the interaction surface. The positions of the mutated residues, V150 and 152, are indicated on each of the subunits. Reprinted from Nature Immunology, with copyright permission from the Nature Publishing Group Hernanz, P., etal. (2004). Identification of amino acid residues crucial for chemokine receptor dimerization. Nature Immunology 5,216-22 i. (See Color Insert.)...

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