Hyperthermophilic l-asparaginase bypasses monomeric intermediates during folding to retain cooperativity and avoid amyloid assembly

In obligate dimeric proteins of hyperthermophilic origin the question whether the native dimer is obtained by association of folded monomers or through concomitant folding and assembly of subunits has intrigued researchers. To find an answer we studied the folding of a dimeric enzyme l-asparaginase from Pyrococcus furiosus (PfA) for which we reported earlier that it unfolds cooperatively without populating folded monomeric intermediates. However, in the present study we report the finding of a folded monomeric intermediate of PfA under acidic condition. This monomer, although inactive, displayed secondary and tertiary structural features identical to the native protein and re-assembled to active dimeric form upon reversal of pH. The monomer is conformationally flexible and thermodynamically and kinetically less stable than the native dimer. Interestingly, when incubated at 60 °C the folded monomer, with exposed ANS-binding hydrophobic surfaces, spontaneously converted to amyloid fibrils. On the basis of our data we propose that PfA directly assembles into a multimeric form perhaps as an evolutionary adaptation to avoid accumulation of aggregation prone monomeric intermediates.