Single-site mutants

Extensive mutagenesis studies have provided information related to the structural integrity, receptor binding region, and residues that are important for IL-1 function. Site-directed mutagenesis (SDM) can create a single-site mutant and its receptor binding and bioactivity values can be calculated. The results... 

When the electrochemistry of the complexes of cytochrome bg with wild-type cytochrome c and several single-site mutants, e.g., Lys-27-Gln, Lys-72-Asp, and Asn-52-Ile, was examined at edge-plane graphite electrodes, a number of differences were observed (30). By comparison with the response in the complex with wild-type cytochrome c, faradaic currents of cytochrome bg were reduced in the complexes with Lys-27-Gln and Asn-52-Ile (Fig. 6a), and were completely abolished in the complex with Lys-72-Asp (Figure 6b). These results suggest that, depending on the site of modification, the binding domain on cytochrome c for the electrode or cytochrome bg can be partially or completely destroyed. 

Figure 3. The segment consisting of residues Cys-192 and His-193 of the 2.8 A resolution structure of a single site mutant of aspartate aminotransferase [23]. Superimposed are the initial structure (dotted lines) obtained by fitting the atomic model to a multiple isomorphous replacement map, the structure obtained after several cycles of rebuilding and restrained least-squares refinement (thick lines), the structure obtained after simulated annealing refinement (thin lines), and the structure obtained after conjugate gradient minimization (dashed lines).

The semiclassical Marcus equation can be applied to electron transfer between spatially fixed and oriented redox centers (26). Values obtained by employing this theoretical framework are in good agreement for both wild type and single-site mutants of Pae azurin (15), thus supporting the applicability of this analysis of the LRET process. The mechanism of intramolecular electron transfer through matrices of biological macromolecules, mainly proteins, has attracted considerable current interest (25). Moreover, the question of whether... 

Biotechnological syntheses of natural proteins or single-site mutants (one amino acid exchanged) occur via appropriate DNA polymers, since each of the 20 natural amino acids is coded by a nucleotide triplet (e.g., AGA codes for arginine). 

Figure 9.3.4 Production of protein single-site mutants by base mismatching of a single nucleic base.

Whether it is the central position proposed for Asp70 in the electrostatic stress mechanism [12] or the role of Lys72 as proton-donor to the carbanion intermediate [24], it is obvious that these two residues are the most important ones in catalysis. Nevertheless, all Asp70 as well as Lys72 single-site mutants of M. thermoautotrophicum ODCase that were generated still retained some enzymatic activity (see below), a clear indication that it is not a sole residue carrying all of the catalytic load [21]. 

Figure 6. CIDNP-diflference spectra (aromatic part) of three single-site mutants of recombinant EcorL. a. [W135A]EcorL (ligand-free) b. [Y106G]EcorL (ligand-free) c. [Y108T]EcorL (ligand-free).

Table 1. Average surface accessibilities (x) and the differences of the surface accessibility areas of CIDNP-sensitive residues derived from molecular dynamics calculations between the wild-type of EcorL (x) and the three single-site mutants [7]. All area values are given in A. Dot density 1 test sphere radius 1.5 A. denotes the site of mutation substituting the respective tyrosine residue with CIDNP-inert alanine.

Figure 7. MD-derived energy minimum structures of wild-type EcorL and single-site mutants, a. wild-type EcorL b. mutant [Y229A]EcorL.

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