MUTATIONAL EFFECTS ON INCLUSION-BODY FORMATION IN THE PERIPLASMIC EXPRESSION OF THE IMMUNOGLOBULIN V-L DOMAIN REI

Background: Inclusion body (IB) formation in bacteria is an important example of protein misassembly, a phenomenon which also includes foldi ng-dependent aggregation in vitro and amyloid deposition in human dise ase. Previous studies of mutational effects in other systems implicate the stability of a folding intermediate-rather than the native state- as playing a key role in IB formation. To contribute to an understandi ng of the comparative biophysics of V-L misassembly in different biolo gical settings, we have studied mutation-dependent periplasmic IB form ation by the V-L domain REI in Escherichia coli. Results: A series of mutants were produced in periplasmic IBs, where, in all cases, the sig nal peptide was removed. In addition, the intradomain disulfide was cl early formed before deposition into IBs. IB formation in these mutants does not correlate with monomer/dimer equilibrium constants, but does correlate with the thermodynamic stability of the native state. Concl usions: The results implicate a late, equilibrium folding intermediate in IB formation, in contrast to the apparent involvement of transient folding intermediates in other IB systems described to date, As equil ibrium unfolding intermediates have also been implicated in light chai n amyloidosis and deposition diseases, IB formation may prove a useful model for these human diseases.