Multi-domain protein folding
Folding is a prerequisite for protein function. Traditionally, protein folding studies have focused on small single domain proteins to understand principles by which a linear polypeptide folds into its functional three-dimensional structure. However, many proteins in extant proteomes are composed of multiple units called domains. Combining multiple domains into a single polypeptide chain is a widely used evolutionary strategy to generate proteins with novel functions, especially in eukaryotic organisms. How these multi-domain proteins fold into their native structures in a timely and efficient way remains poorly understood.
Multi-domain proteins have a high propensity to misfold and aggregate, hampering folding studies using traditional approaches. We have developed single-molecule force spectroscopy with optical tweezers as a platform to manipulate and study the folding and function of large multi-domain proteins. By applying and measuring piconewton forces, and monitoring folding transition with nanometer precision, we can follow the folding of individual protein molecules at high temporal resolution. Our work revealed several novel aspects that are important for understanding the folding of multi-domain proteins.
Co-translational folding. Large proteins begin to fold while the ribosome is still translating the messenger RNAs that encode them. We showed that co-translational folding minimizes misfolding among neighboring domains. The nascent protein interacts with the ribosome as it gradually emerges during synthesis. We showed that interactions with the ribosome can either increase or decrease folding rates, depending on nascent chain length and identity. This finding reconciles well-established, yet seemingly contradictory requirements for co-translational folding: preventing premature folding in emerging domains and promoting timely folding of fully synthesized ones. We are currently studying how variations in the elongation rate (e.g., through the presence of rare codons in natural coding sequences) affect co-translational structure formation.
Chaperone function. Experiments with the five-domain protein elongation factor G (EF-G) revealed that, surprisingly, nascent chain folding does not proceed unidirectionally. Unfolded polypeptide emerging from the ribosome can denature already folded domains, resulting in global misfolding. Strikingly, this unanticipated reversal of folding is effectively blocked by the nascent chain-binding chaperone, trigger factor [Liu et al., 2019, Molecular Cell]. Our experiments revealed a new paradigm for chaperone function that goes beyond the well-established prevention of misfolding among unstructured domains. Unfolded polypeptide is inevitably present during protein synthesis, which might explain the ubiquity of dedicated nascent chain-binding chaperones. Ongoing work is directed at defining operating mechanisms of other chaperones that are important for efficient folding of newly synthesized proteins, such as the Hsp70 and Hsp90 systems.
Domain-domain interactions. Our single-molecule experiments with EF-G revealed that the central domain III is energetically coupled to its C-terminal neighbors, domains IV and V. This energetic coupling of domain stabilities allows domain III to function as a hinge, facilitating large conformational changes in EF-G that are important for its cellular function. However, it also imposes a post-translational folding mechanism that leads to misfolding among the C-terminal domains [Liu et al., 2019, PNAS]. Our experiments thus provide an example of how distinct biological requirements – robust folding and functional flexibility – come into conflict during protein biogenesis. We are currently dissecting the domain interactions in EF-G homologs using equilibrium and non-equilibrium single-molecule measurements.
Nascent chain folding in vivo
Single-molecule in vitro experiments are ideally suited to reveal molecular details of co-translational folding and chaperone interactions. Experiments with live cells are required to capture the complex interplay of the numerous factors that compete for nascent chain access in the crowded environment of the cytosol. We have developed an assay that reports on nascent chain folding in living cells, based on the force-sensing SecM arrest peptide [Goldman et al., 2015, Science]. SecM arrests elongation in cis by interacting with the ribosome exit tunnel. We demonstrated that mechanical force releases SecM arrest, and that folding of a nascent domain in close proximity to the ribosome generates force. Folding just outside the ribosome therefore causes tension on the arrest peptide, resulting in arrest release. Combined with a reporter, this tool allows us to detect “folding waypoints”, i.e. stably folded structures en route to the native state, of nascent polypeptides in vivo.
We have used this approach to monitor nascent chain folding in living cells with a number of candidate proteins and reporters. For instance, we were able to follow folding of the N-terminal G-domain of EF-G using an on-plate colony luminescence assay [Chen et al., 2020, BioRxiv, 2020.04.29.068593]. We are also combining this approach with fluorescence-activated cell sorting to screen through larger libraries of multi-domain proteins. This work complements the biophysical single-molecule studies and provides insights into nascent chain folding in the complex environment of the cytosol.
Protein transport across membranes
The Sec translocon functions as a passageway for regulated protein export from the cytosol across the plasma membrane. It transports substrate proteins ranging from collagen or antibodies in eukaryotes to virulence factors such as protein A or beta-lactamases in bacteria. The Sec system must transport widely heterogeneous polypeptides faithfully and efficiently while maintaining a permeability barrier during translocation to prevent the leakage of ions and breakdown of the proton motive force. The Sec translocon consists of a central heterotrimeric channel that enables passage of unfolded proteins through the bacterial plasma membrane, and an ATPase that drives translocation forward (SecA in bacteria). How the SecA motor utilizes chemical energy from ATP hydrolysis to achieve directed protein transport remains a key unanswered question in biology.
Defining the motor mechanism of SecA. Conflicting models have proposed that the bacterial translocon motor, SecA, either actively generates mechanical force or acts as a ratchet to drive translocation. We are combining biochemical translocation experiments, single-molecule optical tweezers measurements, and mathematical modeling to address this question [Gupta et al., bioRxiv 2020.04.29.066415]. Measuring translocation of DHFR, a protein of tunable stability, with high time resolution and developing a novel kinetic model for data analysis, we found that the rate-limiting step in the process is substrate protein unfolding. We also quantitatively defined the mechanical strength of the DHFR probe using single-molecule optical tweezers experiments. Relating the two measurements indicates that SecA generates mechanical force to actively unfold translocating proteins, arguing for a power-stroke model of SecA function. Combining biochemical and single-molecule measurements has allowed us to define how the SecA motor ensures efficient and robust export of proteins containing stable structure. Ongoing work is aimed at probing the SecA motor mechanisms directly with optical tweezers experiments.