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Correct folding is a prerequisite for the biological activity of most proteins. Folding has largely been studied using in vitro refolding assays with isolated small, robustly folding proteins. A substantial fraction of all cellular proteomes is composed of multidomain proteins that are often not amenable to this approach, and their folding remains poorly understood. These large proteins likely begin to fold during their synthesis by the ribosome, a large molecular machine that translates the genetic code. The ribosome affects how folding proceeds, but the underlying mechanisms remain largely obscure. We have utilized optical tweezers to study the folding of elongation factor G, a multidomain protein composed of five domains. We find that interactions among unfolded domains interfere with productive folding in the full-length protein. The N-terminal G-domain constitutes an independently folding unit that, upon in vitro refolding, adopts two similar states that correspond to the natively folded and a non-native, possibly misfolded structure. The ribosome destabilizes both of these states, suggesting a mechanism by which terminal misfolding into highly stable, non-native structures is avoided. The ribosome may thus directly contribute to efficient folding by modulating the folding of nascent multidomain proteins.

polymorphic transition in which they stretch to three times their original length. In this work we investigate the response of individual T4P monomers and short T4P filaments (10-18 monomers) to external force using the steered molecular dynamics method as implemented in the AMBER molecular dynamics software package. The starting structure for T4P filament simulations is based on the recent cryo-EM structure of the T4P from Neisseria gonorrhoeae in which the helical domain of pilin subunits was found to be partially ''melted.'' Our simulations have been designed to directly test the hypothesis that extension of T4P filaments under force involves additional elongation of the ''melted'' region of the helical domain of each pilin monomer. Specifically, we monitor changes of pilin secondary structure over time under applied force in both monomer and filament simulations. We also determine which pilin-pilin interactions are disrupted in the T4P filament during the initial stages of the force-induced conformational change of this system. Finally, we compare the conformational changes observed for individual pilin subunits under force with the changes observed for pilin subunits within the T4P filament environment. Type IV pilus (T4P) filaments are biological polymers composed of many subunits of a protein called pilin. T4P filaments serve a variety of functions for prokaryotic organisms, including adhesion, cell signaling, and ''twitching'' motility. It is known experimentally that T4P filaments can undergo a largescale polymorphic transition in which they stretch to three times their initial length under hundreds of pN scale forces. Recent cryo-electron microscopy based models of T4P have shed new light on the structure of these filaments, and specifically have revealed the presence of a ''melted'' region of helix within each pilin subunit's alpha-helical domain. This ''melted'' region is thought to contribute to the remarkable elasticity of T4P filaments, however, the nature of the polymorphic transition, and the molecular scale rearrangements of pilin subunits that occur within the T4P filament, cannot be determined from this static experimental data alone. In this work we use the GoMARTINI coarse-grained (CG) model, which allows for the study of conformational changes in MARTINI CG proteins, to investigate the response of long T4P filaments to external forces using a steered molecular dynamics protocol. Simulations are run using the GROMACS software package. The recently solved structure of the T4P filament from Neisseria gonorrhoeae is used as the starting model for CG simulations. Several pulling velocities are explored, including 10, 1 and 0.1 Å /ns. Force versus extension profiles are measured, and the filament Young's modulus, a measure of the elasticity of the T4P filament, is calculated. Conformational changes in T4P filament quaternary structure are monitored, in addition to conformational changes of individual pilin subunits. Finally, in addition to the native filament, several mutants are studied to deter...

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