Manipulation of individual molecules with optical tweezers provides a powerful means of interrogating the structure and folding of proteins. Mechanical force is not only a relevant quantity in ...cellular protein folding and function, but also a convenient parameter for biophysical folding studies. Optical tweezers offer precise control in the force range relevant for protein folding and unfolding, from which single-molecule kinetic and thermodynamic information about these processes can be extracted. In this review, we describe both physical principles and practical aspects of optical tweezers measurements and discuss recent advances in the use of this technique for the study of protein folding. In particular, we describe the characterization of folding energy landscapes at high resolution, studies of structurally complex multidomain proteins, folding in the presence of chaperones, and the ability to investigate real-time cotranslational folding of a polypeptide.
Multi-domain proteins, containing several structural units within a single polypeptide, constitute a large fraction of all proteomes. Co-translational folding is assumed to simplify the ...conformational search problem for large proteins, but the events leading to correctly folded, functional structures remain poorly characterized. Similarly, how the ribosome and molecular chaperones promote efficient folding remains obscure. Using optical tweezers, we have dissected early folding events of nascent elongation factor G, a multi-domain protein that requires chaperones for folding. The ribosome and the chaperone trigger factor reduce inter-domain misfolding, permitting folding of the N-terminal G-domain. Successful completion of this step is a crucial prerequisite for folding of the next domain. Unexpectedly, co-translational folding does not proceed unidirectionally; emerging unfolded polypeptide can denature an already-folded domain. Trigger factor, but not the ribosome, protects against denaturation. The chaperone thus serves a previously unappreciated function, helping multi-domain proteins overcome inherent challenges during co-translational folding.
Display omitted
•How the ribosome modulates nascent chain folding switches during elongation•Sequential domain-wise folding reduces misfolding•Co-translational folding can be reversed by an unexpected unfolding pathway•Protection of folded domains is an unanticipated chaperone function
Liu et al. show that domain-wise folding of nascent proteins can be reversed by denaturing interactions with emerging polypeptide. The chaperone trigger factor blocks denaturation and, together with the ribosome, reduces misfolding. The chaperone thus serves a dual function in promoting efficient folding of multi-domain proteins.
A photoredox-catalyzed procedure for the iodoperfluoroalkylation of styrenes and phenylacetylenes using readily available copper phenanthroline catalyst is reported. In contrast to commonly employed ...Ru(bpy)3Cl2, Ru(phen)3Cl2 or fac-Ir(ppy)3, Cu(dap)2Cl is capable to convert styrenes to the corresponding perfluoroalkyl tagged ethylbenzenes, pointing toward an additional role of the copper catalyst beyond photoinduced electron transfer. An inner sphere catalytic cycle involving Cu(III) intermediates or ligand abstraction from a CuI+ intermediate is proposed.
Proteome complexity has expanded tremendously over evolutionary time, enabling biological diversification. Much of this complexity is achieved by combining a limited set of structural units into long ...polypeptides. This widely used evolutionary strategy poses challenges for folding of the resulting multi-domain proteins. As a consequence, their folding differs from that of small single-domain proteins, which generally fold quickly and reversibly. Co-translational processes and chaperone interactions are important aspects of multi-domain protein folding. In this review, we discuss some of the recent experimental progress toward understanding these processes.
Single-molecule force spectroscopy with optical tweezers has emerged as a powerful tool for dissecting protein folding. The requirement to stably attach “molecular handles” to specific points in the ...protein of interest by preparative biochemical techniques is a limiting factor in applying this methodology, especially for large or unstable proteins that are difficult to produce and isolate. Here, we present a streamlined approach for creating stable and specific attachments using autocatalytic covalent tethering. The high specificity of coupling allowed us to tether ribosome-nascent chain complexes, demonstrating its suitability for investigating complex macromolecular assemblies. We combined this approach with cell-free protein synthesis, providing a facile means of preparing samples for single-molecule force spectroscopy. The workflow eliminates the need for biochemical protein purification during sample preparation for single-molecule measurements, making structurally unstable proteins amenable to investigation by this powerful single-molecule technique. We demonstrate the capabilities of this approach by carrying out pulling experiments with an unstructured domain of elongation factor G that had previously been refractory to analysis. Our approach expands the pool of proteins amenable to folding studies, which should help to reduce existing biases in the currently available set of protein folding models.
All cellular proteins are synthesized by the ribosome, an intricate molecular machine that translates the information of protein coding genes into the amino acid alphabet. The linear polypeptides ...synthesized by the ribosome must generally fold into specific three-dimensional structures to become biologically active. Folding has long been recognized to begin before synthesis is complete. Recently, biochemical and biophysical studies have shed light onto how the ribosome shapes the folding pathways of nascent proteins. Here, we discuss recent progress that is beginning to define the role of the ribosome in the folding of newly synthesized polypeptides.
Display omitted
•Folding is a crucial step in the biogenesis of functional proteins.•Interactions with the ribosome guide nascent polypeptide folding.•Recent experimental work is beginning to shed light on mechanisms by which the ribosome modulates protein folding.
Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive ...interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.
Protein synthesis rates can affect gene expression and the folding and activity of the translation product. Interactions between the nascent polypeptide and the ribosome exit tunnel represent one ...mode of regulating synthesis rates. The SecM protein arrests its own translation, and release of arrest at the translocon has been proposed to occur by mechanical force. Using optical tweezers, we demonstrate that arrest of SecM-stalled ribosomes can indeed be rescued by force alone and that the force needed to release stalling can be generated in vivo by a nascent chain folding near the ribosome tunnel exit. We formulate a kinetic model describing how a protein can regulate its own synthesis by the force generated during folding, tuning ribosome activity to structure acquisition by a nascent polypeptide.
Co‐translational folding and molecular chaperone action are crucial for productive protein folding in the cell, but how these processes shape folding pathways remains largely unknown. We have ...utilized translation arrest peptides (APs) to monitor co‐translational folding in live bacterial cells, combined with single‐molecule optical tweezers experiments, to define the co‐translational folding pathway of the GTPase domain (G‐domain) from E. coli elongation factor G (EF‐G). Surprisingly, the 293 amino acid long domain remains unfolded, without forming stable intermediate structures, until it is fully extruded from the ribosome. The full‐length G‐domain transitions to its stable native structure via obligate folding intermediates both in isolation and while bound to the ribosome. Folding therefore follows a strictly sequential pathway that initiates at the very C‐terminus, which is likely imposed by the structure and topology of the G‐domain from EF‐G. Consequently, folding and synthesis proceed in opposite directions. G‐domains represent a common element in a number of multi‐domain proteins. To determine whether their folding pathways are conserved, we have combined our AP approach with cell sorting and deep sequencing into a method that we term “AP profiling”. Homologous G‐domains exhibited distinct folding patterns that are conserved across distant bacterial species. Individual deletion of the primary nascent chain‐binding chaperones, trigger factor and DnaK (Hsp70), resulted in numerous localized changes to co‐translational folding while preserving overall G‐domain folding, highlighting the functional redundancy of cellular chaperone systems. In summary, we have developed AP profiling as a technique for monitoring co‐translational folding in the native cellular environment with high throughput. Combined with single‐molecule force spectroscopy, AP profiling yields a unique view of co‐translational folding and chaperone function.
Abstract
The Sec translocon moves proteins across lipid bilayers in all cells. The Sec channel enables passage of unfolded proteins through the bacterial plasma membrane, driven by the cytosolic ...ATPase SecA. Whether SecA generates mechanical force to overcome barriers to translocation posed by structured substrate proteins is unknown. Here, we kinetically dissect Sec-dependent translocation by monitoring translocation of a folded substrate protein with tunable stability at high time resolution. We find that substrate unfolding constitutes the rate-limiting step during translocation. Using single-molecule force spectroscopy, we also define the response of the protein to mechanical force. Relating the kinetic and force measurements reveals that SecA generates at least 10 piconewtons of mechanical force to actively unfold translocating proteins, comparable to cellular unfoldases. Combining biochemical and single-molecule measurements thus allows us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.
PDF
