SummaryAt what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins.

Recent experiments carried out in the dense cytoplasm of living cells have highlighted the importance of proteome composition and nonspecific intermolecular interactions in regulating macromolecule diffusion and organization. Despite this, the dependence of diffusion-interaction on physicochemical properties such as the degree of poly-dispersity and the balance between steric repulsion and nonspecific attraction among macromolecules was not systematically addressed. In this work, we study the problem of diffusion-interaction in the bacterial cytoplasm, combining theory and experimental data to build a minimal coarse-grained representation of the cytoplasm, which also includes, for the first time to our knowledge, the nucleoid. With stochastic molecular-dynamics simulations of a virtual cytoplasm we are able to track the single biomolecule motion, sizing from 3 to 80 nm, on submillisecond-long trajectories. We demonstrate that the size dependence of diffusion coefficients, anomalous exponents, and the effective viscosity experienced by biomolecules in the cytoplasm is fine-tuned by the intermolecular interactions. Accounting only for excluded volume in these potentials gives a weaker size-dependence than that expected from experimental data. On the contrary, adding nonspecific attraction in the range of 1-10 thermal energy units produces a stronger variation of the transport properties at growing biopolymer sizes. Normal and anomalous diffusive regimes emerge straightforwardly from the combination of high macromolecular concentration, poly-dispersity, stochasticity, and weak nonspecific interactions. As a result, small biopolymers experience a viscous cytoplasm, while the motion of big ones is jammed because the entanglements produced by the network of interactions and the entropic effects caused by poly-dispersity are stronger.

S2 SI Methods Creation of folding-incompetent domainsTo create destabilized (i.e. non-folding) proteins the same procedure was used to determine an η value that resulted in a native state that was 1.81 kcal/mol higher in free energy than the unfolded state at 310 K. The domain is unfolded 95% of the time at this stability. To ensure that these destabilized proteins were not adopting partially-folded intermediates, all RMSDs were above the threshold value. Rewiring of domainsProteins 1NY8 and 1P9K were rewired to change their topology. To create these rewired mutants, all secondary structural elements in the all-atom structure were fixed in space in their crystallographic configuration, and the unstructured loops were cut and reconnected to change the order of the secondary structural elements along the primary structure. The loop residues were then minimized using the CHARMM22 force-field, during which the secondary structural elements were constrained. The resulting structure was then coarse-grained as previously described, and tuned to the same native-state stability as the wild-type domain.