Protein design and specificity alteration

The alteration of substrate specificity is one of the most challenging goals of protein engineering and protein design. In the case of RNase T1, a target has been to alter the specificity from guanosine in the $G_S^B$-position to adenosine. In early approaches, theoretical structure predictions were used to ``design'' proteins with altered properties[33] by exchanging amino acids in the binding pocket of the enzyme. Being unsuccessful this way[25], so-called semi-rational methods were applied via random mutagenesis of selected amino acids. In a first approach, the amino acids 41 to 46 - the area of substrate recognition (see 1.2.2) - were randomly exchanged to obtain a library of different variants. Apart from a number of enzymes with unchanged specificity, one variant (8/3) could be found with a noticeable increased affinity towards adenosine in the $G$ subsite. However, though this variant had an 5',3'-ApC/5',3'-GpC preference of 0.1 (in contrast to RNase T1 with 2.5 $\times$ 10$^{-5}$), it still preferred guanosine at the $G_S^B$ subsite and had an activity well below the activity of the wild type (1.2 per cent)[35]. Another variant (9/5) from this library characterised by KATJA HÖSCHLER et al. [34] appears to be a promising starting point for a further rational protein modification with altered specificity (RNase T1 RV (sequence altered between amino acid residue 41 and 46 to EFRNWN), RIKO CZAJA, personal communication).