Protein single-site

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

It has been known for some time that tetracyclines are accumulated by bacteria and prevent bacterial protein synthesis (Fig. 4). Furthermore, inhibition of protein synthesis is responsible for the bacteriostatic effect (85). Inhibition of protein synthesis results primarily from dismption of codon-anticodon interaction between tRNA and mRNA so that binding of aminoacyl-tRNA to the ribosomal acceptor (A) site is prevented (85). The precise mechanism is not understood. However, inhibition is likely to result from interaction of the tetracyclines with the 30S ribosomal subunit because these antibiotics are known to bind strongly to a single site on the 30S subunit (85). 

Recently Alan Fersht, Cambridge University, has developed a protein engineering procedure for such studies. The technique is based on investigation of the effects on the energetics of folding of single-site mutations in a protein of known structure. For example, if minimal mutations such as Ala to Gly in the solvent-exposed face of an a helix, destabilize both an intermediate state and the native state, as well as the transition state between them, it is likely that the helix is already fully formed in the intermediate state. If on the other hand the mutations destabilize the native state but do not affect the energy of the intermediate or transition states at all, it is likely that the helix is not formed until after the transition state. 

Trubetskoy, V.S., Narula, J., Khaw, B.A., and Torchilin, V.P. (1993) Chemically optimized antimyosin Fab conjugates with chelating polymers Importance of the nature of the protein-polymer single site covalent bond for biodistribution and infarction localization. Bioconjugate Chem. 4, 251-255. 

In applying this principle to proteins, one would ideally like to modify a protein at one specific site with a number of related, substitution-inert, inorganic redox reagents, and then study the intramolecular electron transfer step as a function of a wide variety of variables (e.g., the redox potential and hydrophobicity of the redox reagent). Such a study is extremely difficult to carry out with large proteins, and none has been reported thus far. We have, however, found out that horseheart cytochrome c is amenable to modification at a single site by the... 

The theory was that the later genomic coamplification would produce a more sfable cell line if performed on cells with a single-site integration rather than those derived from multiple copies even if final protein expression levels were similar, our selected CHO line would possess between 10 and 20 copies of the DNA construct, whereas selecting and coamplifying cells with multiple initial copies would result in a cell line with an order of magnitude of more copies of the construct. This latter condition would increase the chance of unwanted mutation and instability in the cell line. Empirically, our hypothesis seems to have been bom out The CHO line used to produce rituximab and other amplified cell lines have few final copies and are highly stable, even in the absence of MTX. ... 

Thus structural background suggests that the bound cyt c ccp adduct may actually consist of a distribution of structures. In this section, we consider the thermodynamics of binding cyt c and ccp, both for the native proteins, from different species, and proteins incorporating single site replacements, as prepared by site directed mutagenesis. 

The type 1-3 terminology to distinguish different Cu protein active sites remains extremely useful. Sub-groupings are appearing however in all three categories particularly in the case of the binuclear (EPR inactive) type 3 centers. Thus, in the recently determined X-ray crystal structure of ascorbate oxidase the type 3 and type 2 centers are present as a single trimer unit [. A discrete binuclear type 3 center is, however, retained in hemocyanin [6]. 

Substituting these expressions into the equations above yields a new expression that enables the interaction kinetics to be readily modeled for single-site, reversible binding between a protein and a single ligand ... 

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