MODELING OF OLIGOMERIZATION AND FIBRILLOGENESIS OF BETA-2 MICROGLOBULIN MUTANT FORMS

A study has been performed in order to develop the approaches for therapy of amyloidosis using a model object — amyloidogenic protein human beta2-microglobulin (β2m). The aim of the study was to obtain protein variants with amino acid substitutions modulating fibrillogenic properties of β2m which potentially could inhibit fibrillogenesis of the wild-type protein. The study consisted of the two steps: a) computer modelling of β2m associative properties when introducing amino acid substitutions in the primary structure resulting in a change in the degree of molecular symmetry and b) experimental investigation of selected protein variants properties by appropriate genetic constructs design and obtaining the desired recombinant β2m variants. Based on computer calculations virtual variants with double substitutions at the N- and C-terminal regions of the protein molecule have been selected that could potentially provide alterations of monomer the associative properties. These variants have been obtained as recombinant products resulting from expression of genetic constructs specially designed. To obtain the construct with substitutions in the central region of the protein molecule an original double polymerase chain reaction (PCR) method has been developed yielding the intermediate extended primer — a β2m gene fragment with target substitutions which was used as a forward primer in the second PCR. It has been shown that recombinant β2m variants are capable to form fibrils.

amyloidosis, beta2-microglobulin, fibrillogenesis