“rMUP: selection, cloning and expression of three site-directed mutated proteins and preliminary structural characterisation”

MUPs (Major Urinary Proteins) are lipocalins involved in chemical signalling that share a common -barrel structure made of eight antiparallel -strands that is able to accommodate small hydrophobic molecules. A recombinant MUP (rMUP) was cloned, expressed in P. pastoris and the 3D structure determined by NMR (1, 2). To understand the conformational features that underline MUPs biological activity we decided to study various rMUP mutants. Mutation sites were identified based on their influence on functional and structural properties of the protein. To identify these sites we used Circular Dichroism (CD) and fluorescence spectroscopy. Three sites were selected: Cys138, Tyr120 and Tyr84 based on the following data: a) rMUP thermal unfolding is irreversible. We hypothesized that Cys138 could form non-native intramolecular disulphide bonds due to an interchange process with the only SS bridge present. The mutant Cys138Ser has been cloned, expressed and purified. The preliminary structural analysis carried out using CD spectroscopy shows that its thermal denaturation is reversible. b) The structures of rMUP (2) and MUP-I(3), in solution, show that Tyr120 and Tyr84 point into the hydrophobic pocket and interact with the bound pheromone 2-sec-buthyl-thyazoline. We carried out a CD study on rMUP confirming that indeed the binding of the fluorescent ligand 2-naphtol produces a perturbation of the Tyr signals. To verify the importance of these residues for the binding affinity, mutants, where Tyr120 and Tyr84 will be substituted either with Ala or Phe, are being prepared. The strategy of cloning and expression of these mutants will be discussed. Overall we believe that studies of mutated proteins carried out under different experimental conditions and using spectroscopic techniques are essential to further the comprehension of the structural, dynamic and functional properties of lipocalins. 1) Ferrari E. et al. FEBS Lett. 1997, 401, 73-77; 2) Lücke C. et al. Eur. J. Biochem., 1999, 266, 1210-1218; 3) Zidek et al. Biochemistry 1999, 38, 9850-9861