A structural genomics pilot project : methods and applications

Sammanfattning: With the availability of many completely sequenced genomes, scientific research has shifted from genes to the products of the genes, the proteins. Structural genomics groups have been established worldwide, with the objective of determining protein structures on a genome-wide scale. New methods for protein production and structural determination have become necessary.Two methods for high throughput analysis of proteins are presented in the first part of this thesis. The first method is the thermofluor method, which presents a fast way to identify stabilizing conditions for a particular protein. It was shown that the addition of a stabilizing additive, identified with the thermofluor method, significantly increased the likelihood of growing protein crystals. The second method presented in this thesis provides a fast and robust way to detect metal containing proteins.The second part of this thesis describes the crystal structures of two RNA modifying enzymes, the pseudouridine synthase TruD and the RNA m5C methyltransferase YebU. The catalytic domain of TruD was shown to bear remarkable structural similarity to the other pseudouridine families despite a lack of sequence similarity. In addition to the catalytic domain, the structure of TruD also contained an insertion domain with a novel fold.YebU was also found have two structurally distinct domains. The N-terminal catalytic domain has a high structural similarity to other RNA m5C methyltransferases. The C-terminal domain was revealed to be a so-called PUA domain, which had not been predicted by previous sequence alignments.