RICERCA ITALIANA     

Research program

Identification of folding and misfolding determinants by site-directed mutagenesis.

University Co-ordinator

Università degli Studi di FIRENZE - SCIENZE BIOCHIMICHE - ()

Research Unit Leader

Fabrizio Chiti

Description

This research unit (UR) has a long standing experience in the utilisation of protein engineering as a means of gaining insight into the processes of both folding and aggregation. The first duty of this UR will be the design of specific mutations for the production of several protein variants, each of which will have one single substituted residue. We will design a number of mutations that are useful to study both the processes of folding and aggregation. The mutations will be chosen on the basis of a number of factors: the need to be as conservative as possible, to cover the full length of the b2-m sequence and different structural regions of the native protein, to involve both solvent-exposed residues and others that are buried in the core and, most importantly, to be able to affect the folding and aggregation rates of b2-m so that the involvement of the mutated residues on the two process can be evaluated. The list of chosen mutations will be handed in to the UR of Pavia (M. Stoppini) so that the protein variants can be expressed and purified. The UR in Pavia will then make the mutants available to all the Urs involved in the present project.
A second task of this UR will consist in the investigation of the thermodynamic and kinetic parameters of the folding and unfolding reaction of b2-m. The mutants will be received in a purified form by the unit of Pavia (M. Stoppini). The conformational stabilities of the mutants will be determined by acquiring equilibrium GdnHCl-induced unfolding curves using intrinsic fluorescence as a spectroscopic probe to follow unfolding. These curves, acquired for each mutant and for the wild-type protein, will allow the free energy change of unfolding to be determined for each protein variant. A fluorimeter, available in this UR, will be used for this purpose. A circular dichroism device will also be utilised if fluorescence turns out to be an unsuitable probe for some of the mutants.
The folding and unfolding rate constants will be determined for each protein variant at different Gdn-HCl concentrations using a sopped-flow device coupled to a fluorescence detection system. A stopped-flow device coupled to circular dichroism detection system will also be used if fluorescence turns out to be an unsuitable probe for some of the mutants. The kinetic and thermodynamic data of the mutants and of the wild-type protein will be combined to determine the phi-values of all the mutated residues at the level of both the folding transition state and of the intermediate state that remains populated after the folding reaction has reached equilibrium.
As a third approach this UR will use all the algorithms that have been published so far to determine the theoretical aggregation rates and/or propensities expected for each mutant. The comparison and the possible deviations between the theoretical and experimental values (the latter obtained and communicated by the UR of Pavia and Genova, headed by M. Stoppini and A. Relini, respectively) will reveal not just the regions of the sequence or residues that promote aggregation, but also whether the mechanism of aggregation involves the unfolding or refolding of a portion of structure or additional steps of non-amyloid aggregation prior to the establishment of amyloid structures. Softwares will be produce to run the algorithm on a computer.
Finally, given the expertise of this UR in protein folding, protein aggregation and protein engineering, we will try hard to collect the data from all the other units and to use them to determine the residue-specific mechanisms of aggregation and folding of b2-m.