Jean Chatellier

The substrate binding domain of DnaK facilitates slow protein refolding

We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states. The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution. The effects of DnaK384-638 on the reactivation of β-galactosidase and luciferase were examined at low substrate concentration and low temperature, conditions in which the folding is significantly slow (several days) but the reactivation yield is higher than those in ordinary refolding conditions. In the presence of DnaK384-638, the maximum yield of active β-galactosidase was improved from 45% to 65% after a 48-h refolding reaction. Spectroscopic experiments showed that DnaK384-638 bound to partially structured monomers of β-galactosidase and consequently suppressed aggregation. DnaK384-638 accelerated the refolding of luciferase to attain equilibrium in 8 h. On the other hand, DnaK386-561, which has no affinity for the substrate, had no chaperone activity for the reactivation of these proteins. These results indicate that the substrate binding of DnaK384-638 facilitates slow protein refolding.

Interdomain interactions within the gene 3 protein of filamentous phage

Infection of Escherichia coli by filamentous phage fd is mediated by the phage gene 3 protein (g3p). The g3p consists of three domains (g3p‐D1, D2 and D3) linked by flexible glycine‐rich linkers. All three domains are indispensable for phage infectivity; the g3p‐D1 domain binds to the TolA receptor presumably at the inner face of the outer membrane, the g3p‐D2 domain to the F‐pilus and the g3p‐D3 domain anchors g3p to the phage coat. The N‐terminal domains g3p‐D1 and D2 interact with each other; this interaction is abrogated by binding of g3p‐D2 to the F‐pilus leading to the release of g3p‐D1 to bind to TolA. Here, using phages with deletions in g3p, we have discovered a specific interaction between the two N‐terminal domains and g3p‐D3, the C‐terminal domain of g3p. We propose that these interdomain interactions within g3p lead to a compact and stable organisation when displayed on the phage tip, but that during infection, this compact state must be unraveled.