Trigger factor chaperone acts as a mechanical foldase

Abstract

Proteins fold under mechanical forces in a number of biological processes, ranging from muscle contraction to co-translational folding. As force hinders the folding transition, chaperones must play a role in this scenario, although their influence on protein folding under force has not been directly monitored yet. Here, we introduce single-molecule magnetic tweezers to study the folding dynamics of protein L in presence of the prototypical molecular chaperone trigger factor over the range of physiological forces (4-10 pN). Our results show that trigger factor increases prominently the probability of folding against force and accelerates the refolding kinetics. Moreover, we find that trigger factor catalyzes the folding reaction in a force-dependent manner; as the force increases, higher concentrations of trigger factor are needed to rescue folding. We propose that chaperones such as trigger factor can work as foldases under force, a mechanism which could be of relevance for several physiological processes.Proteins fold under mechanical force during co-translational folding at the ribosome. Here, the authors use single molecule magnetic tweezers to study the influence of chaperones on protein folding and show that the ribosomal chaperone trigger factor acts as a mechanical foldase by promoting protein folding under force.

Fig. 1

Trigger factor and the folding dynamics of protein L. a Schematics of the magnetic tweezers experiment, showing the octamer of protein L tethered between a glass coverslip and the paramagnetic bead. The force is applied by changing the separation of the permanent magnets and the bead. b Dynamics of protein L octamer at three different forces with (red) and without (black) TF. First, the protein is fully unfolded by a fingerprint pulse, where the eight unfolding events are identified as eight length steps. Second, a refolding pulse is set at a lower force (4.3, 7.4 and 11.9 pN, from bottom to top). At 4.3 pN (faint color, lowest length) all domains are able to fold by themselves, leading to a maximum probability of folding. Therefore, TF does not do any significant effect. At 11.9 pN (faint color, highest length), the protein is not able to refold (0 probability of folding) and TF does not either affect the probability of folding. In the intermediate force range (7.4 pN, solid colors) TF greatly increases the probability of folding, reflected in a higher number of folded domains

Fig. 2

Effect of trigger factor on the folding properties of protein L under force. a Representative force-clamp magnetic tweezers trajectory of a protein L octamer in presence of 500 μM TF with the monitored properties highlighted. After the unfolding pulse, the molecule is set at a constant force (8.1 pN in this case), relaxing to an equilibrium state. The kinetic properties are reflected in the first passage time (FPT), time taken to, starting with all domains unfolded, fold the eight domains for the first time (time between arrows, highlighted). In equilibrium, the molecule experiences several folding-unfolding transitions, reflected in different residence times at each folding state (labeled as the number of folded domains), marked with the dashed blue lines. The folding probability is calculated by monitoring the fraction of time spent on each of the states (black bars). b Mean-FPT for total refolding in presence (red) and absence (black) of TF. TF modulates the folding kinetics by speeding up the time needed to refold all eight domains. Above 7.4 pN, a protein L octamer needs over 1000 s to fold completely, while TF accelerates folding over an order of magnitude. Each data point represents the average of more than ten experiments. Error bars represent s.e.m. c Folding probability in presence (red) and absence (black) of TF. The presence of the molecular chaperone TF increases considerably the folding probability over the range from 7 to 10 pN. The inset shows the relative increment, reaching up to 40%. Data points are calculated as described above, using >3000 s, and over more than three molecules per force. Errors bars are s.e.m

Fig. 3

Titration curve describing the force-dependent effect of the trigger factor concentration on the folding probability of protein L. We monitor the change in the folding probability of protein L at increasing TF concentration for different forces. The sigmoidal dependence of the folding probability with the TF concentration reveals the cooperative effect of TF on protein L under force. Inset shows the half maximal concentration K ^eff _1/2, showing a strong non-linear dependence of the TF effective affinity with the force. Data points are calculated as described above, using >3000 s, and over more than three molecules per force. Errors bars are s.e.m

Fig. 4

Model for trigger factor-assisted force transmission through a molecular pore. a A polypeptide chain exiting a molecular tunnel is under some tension due to the confined geometry (left). When the protein folds in the edge of the tunnel (right), it pulls out a fraction of the polypeptide in the channel (purple fragment), increasing the mechanical tension and hampering the folding transition. b When a polymer is confined in a tube of fixed length L _tunnel is subject to an effective force, which depends on its contour length L _contour. This force can be approximately modeled by a freely jointed chain (FJC) model, where the end-to-end distance is fixed to L _tunnel and the force depends on the length of the confined polymer L _contour (blue line). Accordingly, the folding probability with (red) and without (black) TF of a polypeptide chain in such scenario depends on L _contour. c Protein folding generates shortening which increases the tension of the polymer, transmitting a force through the tunnel. We can estimate this expected force as the product between the force generated by the confined polymer and the probability of folding at such length L _contour. This is calculated with (red) and without (black) TF revealing that there is an optimal length of the confined polymer, between 3 and 4 times the length of the tunnel. Shorter polymers would induce large tensions that would avoid protein folding, so the expected force is zero. Longer polymers would lead to small values of the expected force. In the optimal region, TF boosts the transmitted force by almost 4 pN (purple)