Highlights d How the ribosome modulates nascent chain folding switches during elongation d Sequential domain-wise folding reduces misfolding d Co-translational folding can be reversed by an unexpected unfolding pathway d Protection of folded domains is an unanticipated chaperone function

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.

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The coupling of protein synthesis and folding is a crucial yet poorly understood aspect of cellular protein folding. Over the past few years, it has become possible to experimentally follow and define protein folding on the ribosome, revealing principles that shape co‐translational folding and distinguish it from refolding in solution. Here, we highlight some of these recent findings from biochemical and biophysical studies and their potential significance for cellular protein biogenesis. In particular, we focus on nascent chain interactions with the ribosome, interactions within the nascent protein, modulation of translation elongation rates, and the role of mechanical force that accompanies nascent protein folding. The ability to obtain mechanistic insight in molecular detail has set the stage for exploring the intricate process of nascent protein folding. We believe that the aspects discussed here will be generally important for understanding how protein synthesis and folding are coupled and regulated.

The pathological accumulation of cholesterol is a signature feature of Niemann–Pick type C (NPC) disease, in which excessive lipid levels induce Purkinje cell death in the cerebellum. NPC1 encodes a lysosomal cholesterol-binding protein, and mutations in NPC1 drive cholesterol accumulation in late endosomes and lysosomes (LE/Ls). However, the fundamental role of NPC proteins in LE/L cholesterol transport remains unclear. Here, we demonstrate that NPC1 mutations impair the projection of cholesterol-containing membrane tubules from the surface of LE/Ls. A proteomic survey of purified LE/Ls identified StARD9 as a novel lysosomal kinesin responsible for LE/L tubulation. StARD9 contains an N-terminal kinesin domain, a C-terminal StART domain, and a dileucine signal shared with other lysosome-associated membrane proteins. Depletion of StARD9 disrupts LE/L tubulation, paralyzes bidirectional LE/L motility and induces accumulation of cholesterol in LE/Ls. Finally, a novel StARD9 knock-out mouse recapitulates the progressive loss of Purkinje cells in the cerebellum. Together, these studies identify StARD9 as a microtubule motor protein responsible for LE/L tubulation and provide support for a novel model of LE/L cholesterol transport that becomes impaired in NPC disease.

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Multi‐domain proteins – constituting a large group in all proteomes – often require help from molecular chaperones to fold productively, even before the ribosome has finished their synthesis. The mechanisms underlying chaperone function remain poorly understood. We have used optical tweezers to study the folding of elongation factor G (EF‐G), a model multi‐domain protein, as it emerges from the ribosome. We find that the N‐terminal G‐domain in nascent EF‐G polypeptides folds robustly. The following domain II, in contrast, fails to fold efficiently. Strikingly, interactions with the unfolded domain II convert the natively folded G domain to a non‐native state. This non‐native state readily unfolds, and the two unfolded domains subsequently form misfolded states, preventing productive folding. Both the conversion of natively folded domains and non‐productive interactions among unfolded domains are efficiently prevented by the ribosome‐binding chaperone trigger factor. Thus, our single‐molecule measurements of multi‐domain protein folding reveal an unexpected role for the chaperone: It protects already folded domains against denaturation resulting from interactions with parts of the nascent polypeptide that are not folded yet. Previous studies had implicated trigger factor in guiding the folding of individual domains, and interactions among domains had been neglected. Avoiding early folding defects is crucial, since they can propagate and result in misfolding of the entire protein. Our experiments define the folding pathway for a complex multi‐domain protein and shed light on the molecular mechanism employed by molecular chaperones to ensure productive and efficient folding.Support or Funding InformationCMK acknowledges support by the Pew Scholars Program in the Biomedical Sciences and the National Institute of General Medical Sciences (1R01GM121567)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.