A kinetic reaction trap

In order to find our more about the folding of pilus subunits, unfolded FimG was exposed to a mixture of the chaperone FimC and native FimF. The presence of FimF should clarify whether the unfolded subunit FimG will bind to the donor strand of a free chaperone or rather to another chaperone-bound subunit. The results were clear: After less than 3 minutes, FimG bound quantitatively to FimC. FimG then slowly dissociated from FimC and formed a complex with FimF. After 62 hours, only 0.6% of FimG was in complex with FimC - the rest was bound to FimF.

Complex FimG-FimF is thermodynamically more stable than FimG-FimC. A energy difference of at least 17 kJ/mol was calculated based on the final concentrations of these two complexes. Hence, it seems that FimC acts as a kinetic reaction trap preventing premature subunit assembly in the periplasm before the subunit is delivered to the usher FimD. At the same time, energy is preserved that may be used to promote final pilus assemby.

One hundred times faster

In vivo translocation of pilus subunits into the periplasm and incorporation into the growing pilus occurs within seconds. However, spontaneous refolding of e.g. subunit FimG is by far a slower process taking minutes. FimC accelarates this folding process about one hunderd times. This was shown by stopped-flow tryptophan fluorescence in an assay with FimG and FimC. Complementing experiments proved that indeed native FimG was formed and not a folding intermediate. Only FimC is able to accelerate the folding rate, but not the isolated donor strand of FimC alone, indicating that additional contacts between FimC and the subunit are required for the observed effect. The presence of complete FimC is hence a prerequisite for efficient pilus formation by overcoming the kinetic bottleneck of subunit folding in the periplasm.

Interestingly, classical chaperones affect folding eficiency, but do not increase the folding rate. The only other known exception is the GroEL/GroES cavity, that increases the folding rates of some substrates four-fold. However, a completely different reaction mechanism is likely for FimC. It seems that FimC may transiently become part of the tertiary structure of its substrate. Fim C thus represents a new type of protein-folding catalyst that either accelerates certain rate-limiting steps along the folding pathway of pilus subunits of even changes the folding mechanism of its substrates entirely. Nevertheless, FimC is a typical chaperone as it binds to non-assembled substrates and inhibits non-specific aggregation of subunits.