The question of protein design can be phrased as: """"""""What three- dimensional structure is compatible with this amino-acid sequence?"""""""" A more tractable problem is the undesign question: """"""""What sequences are compatible with this structure?"""""""", this latter problem will be our major focus. Our approach, however, differs from that of others: not only shall we do model-building studies but also we will test our predictions by creating chimaeric proteins where 1/8th of the structure is from a """"""""foreign"""""""" protein and 7/8ths is from the protein under examination. We will focus exclusively on the folding of a particular protein domain - the parallel-stranded alpha beta-barrel - expecting that the insights gained here will have general validity. The test system will be muconate lactonising enzyme (MLE), a catabolic enzyme from Pseudomonas putida. It contains an alpha beta-barrel domain: eight parallel beta-strands linked by eight alpha-helices antiparallel to the beta-strands. This regular structure lends itself to the question: """"""""Can we substitute one alpha beta-segment with another?"""""""" Using sequences based on alpha beta-segments from alpha beta-barrel proteins which show no sequence similarity with MLE, we will substitute DNA coding for various alph beta-segments in MLE with DNA coding for new, artificial alpha beta-segments. We will then measure the activity of the chimaeric enzymes, crystallise them and solve their structure. Were they still to fold correctly, we would have shown that the folding of these can be divided into two somewhat independent components: the folding of the alpha-helix onto the beta-strand, and the folding of the supersecondary alpha beta- segments into the full alpha beta-barrel. To understand the structural basis of the enzyme mechanism of MLE, we plan to solve the structure of the muconate lactonising enzyme- substrate cocrystals. We also plan to crystallise and solve the structure of beta-carboxymuconate lactonising enzyme, because it is believed that this enzyme - even though it catalyses a similar reaction to that of MLE in a parallel pathway - is not related by divergent evolution of MLE.
