Synthesis and biochemical characterization of obligatory dimers of the sugar non-specific nuclease from Serratia marcescens using specifically designed bismaleimidoalkanes as SH-specific crosslinking reagents

Abstract

The genetically engineered S140C variant of the homodimeric nuclease from Serratia marcescens was crosslinked across the dimer interface at the Cys 140 residues using bifunctional SH-specific 1,1'-alkanediyl-bis-pyrrole-2,5-diones of different lengths. These bismaleimidoalkanes were synthesized by the condensation of n-alkyldiamines with maleic anhydride and subsequent cyclization with acetic anhydride and sodium acetate. Bismaleimidohexane (BMH) which gave the best crosslinking yield was used to produce in preparative amounts crosslinked Serratia nuclease. The crosslinked protein has the same secondary structure and exhibits the same guanidinium chloride unfolding behavior as the wild type enzyme or the non-covalently linked S 140C variant. In contrast, in thermal unfolding experiments the crosslinked dimer behaves differently from the wild type enzyme or the non-covalently linked S140C variant. CD-spectra recorded during temperature rise showed only minor changes of the secondary structure composition for the wild type enzyme and the non-covalently linked S140C variant, whereas in the case of the crosslinked S140C dimer a distinct increase of the CD effect was observed corresponding to an increase in alpha-helix. Our results demonstrate that bismaleimidoalkanes are very well suited to covalently link subunits of proteins, provided suitably located cysteine residues are present.