Ribosome-bound Nascent Chain Research Articles

Capturing Membrane Protein Ribosome Nascent Chain Complexes in a Native-like Environment for Co-translational Studies.

Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo-generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene–maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.

Trigger Factor-Induced Nascent Chain Dynamics Changes Suggest Two Different Chaperone-Nascent Chain Interactions during Translation

Protein biogenesis is poorly understood due to the ribosome that perturbs measurement attempted on the ribosome-bound nascent chain (RNC). Investigating nascent chain dynamics may provide invaluable insight into the co-translational processes such as structure formation or interaction with a chaperone [e.g., the bacterial trigger factor (TF)]. In this study, we aim to establish a platform for studying nascent chain dynamics by exploring the local environment near the fluorescent dye on site-specifically labeled RNCs with time-resolved fluorescence anisotropy. To prepare a quantitative model of fluorescence depolarization, we utilized intrinsically disordered protein bound to ribosome, which helped us couple the sub-nanosecond depolarization with the motion of the nascent chain backbone. This was consistent with zinc-finger-domain-containing RNCs, where the extent of sub-nanosecond motion decreased upon the addition of zinc when the fluorophore was in close proximity of the domain. After the characterization of disordered nascent chain dynamics, we investigated the synthesis of a model cytosolic protein, Entner–Doudoroff aldolase, labeled at different sites during various stages of translation. Depending on the stage of translation, the addition of the TF to the nascent chain led to two different responses in the nascent chain dynamics serendipitously, suggesting steric hindrance between the nascent chain and the chaperone as a mechanism for TF dissociation from the ribosome during translation. Overall, our study demonstrates the possible use of site-specific labeling and time-resolved anisotropy to gain insight on chaperone binding event at various stages of translation and hints on TF co-translational mechanism.

A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy.

During biosynthesis on the ribosome, an elongating nascent polypeptide chain can begin to fold, in a process that is central to all living systems. Detailed structural studies of co-translational protein folding are now beginning to emerge; such studies were previously limited, at least in part, by the inherently dynamic nature of emerging nascent chains, which precluded most structural techniques. NMR spectroscopy is able to provide atomic-resolution information for ribosome-nascent chain complexes (RNCs), but it requires large quantities (≥10 mg) of homogeneous, isotopically labeled RNCs. Further challenges include limited sample working concentration and stability of the RNC sample (which contribute to weak NMR signals) and resonance broadening caused by attachment to the large (2.4-MDa) ribosomal complex. Here, we present a strategy to generate isotopically labeled RNCs in Escherichia coli that are suitable for NMR studies. Uniform translational arrest of the nascent chains is achieved using a stalling motif, and isotopically labeled RNCs are produced at high yield using high-cell-density E. coli growth conditions. Homogeneous RNCs are isolated by combining metal affinity chromatography (to isolate ribosome-bound species) with sucrose density centrifugation (to recover intact 70S monosomes). Sensitivity-optimized NMR spectroscopy is then applied to the RNCs, combined with a suite of parallel NMR and biochemical analyses to cross-validate their integrity, including RNC-optimized NMR diffusion measurements to report on ribosome attachment in situ. Comparative NMR studies of RNCs with the analogous isolated proteins permit a high-resolution description of the structure and dynamics of a nascent chain during its progressive biosynthesis on the ribosome.

Investigation of SecY protein‐translocation channel in action using a novel in vivo tool (LB198)

Many proteins are targeted by signal sequences to a conserved protein‐conducting channel, formed by the prokaryotic SecY or eukaryotic Sec61 complex, and are translocated across the membrane. The channel forms an hourglass‐shaped pore with a constriction in the middle across the membrane. The channel also has a lateral gate that can open toward the lipid phase. During initiation of translocation, a ribosome‐nascent chain complex binds to the SecY/Sec61 channel, resulting in insertion of the nascent chain. To study the SecY channel engaged in translocation, we developed a novel method generating defined translocation intermediate complexes in E. coli cells. Using this tool, we addressed how the channel is opened by a ribosome‐bound nascent chain and transports large polypeptides and yet prevents passage of small molecules. We determined structures of inactive and active ribosome‐channel complexes with cryo‐electron microscopy. These structures demonstrate that the nascent chain opens the channel through mostly rigid body movements of the two halves of SecY. The polypeptide inserts as a loop into the channel, with the hydrophobic region of the signal sequence intercalated into the open lateral gate. Biochemical analysis shows that the pore residues of SecY form a gasket‐like seal around the translocating polypeptide chain, preventing the permeation of small molecules. This work was supported by HHMI and NIH.

Reorientation of the first signal-anchor sequence during potassium channel biogenesis at the Sec61 complex

The majority of the polytopic proteins that are synthesized at the ER (endoplasmic reticulum) are integrated co-translationally via the Sec61 translocon, which provides lateral access for their hydrophobic TMs (transmembrane regions) to the phospholipid bilayer. A prolonged association between TMs of the potassium channel subunit, TASK-1 [TWIK (tandem-pore weak inwardly rectifying potassium channel)-related acid-sensitive potassium channel 1], and the Sec61 complex suggests that the ER translocon co-ordinates the folding/assembly of the TMs present in the nascent chain. The N-terminus of both TASK-1 and Kcv (potassium channel protein of chlorella virus), another potassium channel subunit of viral origin, has access to the N-glycosylation machinery located in the ER lumen, indicating that the Sec61 complex can accommodate multiple arrangements/orientations of TMs within the nascent chain, both in vitro and in vivo. Hence the ER translocon can provide the ribosome-bound nascent chain with a dynamic environment in which it can explore a range of different conformations en route to its correct transmembrane topology and final native structure.

Mechanisms in Co-Translational Protein Folding Elucidated using Single Molecule FRET

In nature, protein folding mechanisms are determined by both amino acid sequence AND physiological context. Inside living cells the transient and vectorial nature of protein synthesis, the presence of molecular chaperones, and macromolecular crowding can each have a profound effect on the paths nascent chains take en route to their native states. This adds a daunting level of complexity to the study of in-vivo protein folding mechanisms. To begin to address these issues we have developed methods which enable the detection of ribosome-bound nascent chain (RNC) conformational distributions and dynamics using single molecule fluorescence resonance energy transfer. Our approach employs a purified and reconstituted E. coliin-vitro translation system to generate stalled RNCs, the site-specific incorporation of chemically-reactive alkyne-bearing unnatural amino acid tags into nascent chains, and post-translational bioconjugation of pairs of single-molecule donor and acceptor fluorophores to the RNCs using a highly-efficient ligand accelerated copper-click chemistry reaction. We apply our novel RNC labeling approach to explore the role of helix stabilization by and propagation from the exit tunnel of the ribosome as a general means by which the ribosome may direct the co-translational folding of nascent polypeptides.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The cell-free integration of a polytopic mitochondrial membrane protein into liposomes occurs cotranslationally and in a lipid-dependent manner.

The ADP/ATP Carrier (AAC) is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.

Monitoring cotranslational protein folding in mammalian cells at codon resolution

How the ribosome-bound nascent chain folds to assume its functional tertiary structure remains a central puzzle in biology. In contrast to refolding of a denatured protein, cotranslational folding is complicated by the vectorial nature of nascent chains, the frequent ribosome pausing, and the cellular crowdedness. Here, we present a strategy called folding-associated cotranslational sequencing that enables monitoring of the folding competency of nascent chains during elongation at codon resolution. By using an engineered multidomain fusion protein, we demonstrate an efficient cotranslational folding immediately after the emergence of the full domain sequence. We also apply folding-associated cotranslational sequencing to track cotranslational folding of hemagglutinin in influenza A virus-infected cells. In contrast to sequential formation of distinct epitopes, the receptor binding domain of hemagglutinin follows a global folding route by displaying two epitopes simultaneously when the full sequence is available. Our results provide direct evidence of domain-wise global folding that occurs cotranslationally in mammalian cells.

Nuclear translation visualized by ribosome-bound nascent chain puromycylation

Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.

Competitive Binding of the SecA ATPase and Ribosomes to the SecYEG Translocon

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.

Confined dynamics of a ribosome-bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions

Ellis JP, Culviner PH, Cavagnero S. (2009) Protein Sci 18:2003–2015. The ribosome-bound nascent chain denoted as PIR37 should be listed as PIR33, throughout the manuscript. In addition, some order parameters reported in Table 2 need to be corrected to the values shown in bold in the updated Table 2 below. The corrections listed here do not affect any of the reported cone semi-angles, rotational correlation times, result interpretation nor conclusions from this study.

Probing Side-Chain Dynamics of a Ribosome-Bound Nascent Chain Using Methyl NMR Spectroscopy

We report here the use of methyl NMR spectroscopy with a selective-excitation pulsing scheme to extract structural information about a ribosome-bound nascent chain, a complex with a molecular weight of more than 2 MDa and a sample concentration in the micromolar range. The carbon chemical shifts of methyl groups are particularly sensitive to the development of the tertiary structure of a protein it folds, and crucially for systems that are at the limit of acceptable signal-to-noise-ratios, methyl group spectroscopy has higher sensitivity than does backbone amide group-based NMR spectroscopy. Comparison of the side-chain methyl correlations of the ribosome-bound nascent chain to previously obtained backbone amide correlations reveals dynamical perturbations within the hydrophobic core of the folded domain, which are attributed to motional restriction of the nascent chain as a result of ribosome attachment. Methyl NMR spectroscopy therefore provides improved spectral quality and complementary structural information to that of the amide groups and hence promises to provide a greatly enhanced understanding of the molecular basis of cotranslational folding at atomic resolution.

Structure, Dynamics and Folding of an Immunoglobulin Domain of the Gelation Factor (ABP-120) from Dictyostelium discoideum

We have carried out a detailed structural and dynamical characterisation of the isolated fifth repeat of the gelation factor (ABP-120) from Dictyostelium discoideum (ddFLN5) by NMR spectroscopy to provide a basis for studies of co-translational folding on the ribosome of this immunoglobulin-like domain. The isolated ddFLN5 can fold autonomously in solution into a structure that resembles very closely the crystal structure of the domain in a construct in which the adjacent sixth repeat (ddFLN6) is covalently linked to its C-terminus in tandem but deviates locally from a second crystal structure in which ddFLN5 is flanked by ddFLN4 and ddFLN6 at both N- and C-termini. Conformational fluctuations were observed via 15N relaxation methods and are primarily localised in the interstrand loops that encompass the C-terminal hemisphere. These fluctuations are distinct in location from the region where line broadening is observed in ddFLN5 when attached to the ribosome as part of a nascent chain. This observation supports the conclusion that the broadening is associated with interactions with the ribosome surface [Hsu, S. T. D., Fucini, P., Cabrita, L. D., Launay, H., Dobson, C. M. & Christodoulou, J. (2007). Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy. Proc. Natl. Acad. Sci. USA, 104, 16516–16521]. The unfolding of ddFLN5 induced by high concentrations of urea shows a low population of a folding intermediate, as inferred from an intensity-based analysis, a finding that differs from that of ddFLN5 as a ribosome-bound nascent chain. These results suggest that interesting differences in detail may exist between the structure of the domain in isolation and when linked to the ribosome and between protein folding in vitro and the folding of a nascent chain as it emerges from the ribosome.

A signal-anchor sequence stimulates signal recognition particle binding to ribosomes from inside the exit tunnel

Sorting of eukaryotic membrane and secretory proteins depends on recognition of ribosome-bound nascent chain signal sequences by the signal recognition particle (SRP). The current model suggests that the SRP cycle is initiated when a signal sequence emerges from the ribosomal tunnel and binds to SRP. Then elongation is slowed until the SRP-bound ribosome-nascent chain complex (RNC) is targeted to the SRP receptor in the endoplasmic reticulum (ER) membrane. The RNC is then transferred to the translocon, SRP is released, and translation resumes. Because RNCs do not target to the translocon efficiently if nascent chains become too long, the window for SRP to identify its substrates is short. We now show that a transmembrane signal-anchor sequence (SA) significantly enhances binding of SRP to RNCs even before the SA emerges from the ribosomal tunnel. In this mode, SRP does not contact the SA directly but is in close proximity to the portion of the nascent polypeptide that has already left the ribosomal tunnel. Early recruitment of SRP provides a mechanism to expand the window for substrate identification. We suggest that the dynamics of the SRP-ribosome interaction is affected not only by the direct binding of SRP to an exposed signal sequence but also by properties of the translating ribosome that are triggered from within the tunnel.

Protein Translocation Across the Bacterial Cytoplasmic Membrane

About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they are targeted to the Sec translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The Sec translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.

Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy.

Protein folding in living cells is inherently coupled to protein synthesis and chain elongation. There is considerable evidence that some nascent chains fold into their native structures in a cotranslational manner before release from the ribosome, but, despite its importance, a detailed description of such a process at the atomic level remains elusive. We show here at a residue-specific level that a nascent protein chain can reach its native tertiary structure on the ribosome. By generating translation-arrested ribosomes in which the newly synthesized polypeptide chain is selectively (13)C/(15)N-labeled, we observe, using ultrafast NMR techniques, a large number of resonances of a ribosome-bound nascent chain complex corresponding to a pair of C-terminally truncated immunoglobulin (Ig) domains. Analysis of these spectra reveals that the nascent chain adopts a structure in which a native-like N-terminal Ig domain is tethered to the ribosome by a largely unfolded and highly flexible C-terminal domain. Selective broadening of resonances for a group of residues that are colocalized in the structure demonstrates that there are specific but transient interactions between the ribosome and the N-terminal region of the folded Ig domain. These findings represent a step toward a detailed structural understanding of the cellular processes of cotranslational folding.

Cotranslational Folding of Globin

Globin synthesis in a wheat germ cell-free translation system was performed in the presence of [3H]hemin and [35S]methionine to determine the minimal length of the nascent ribosome-bound globin chain capable of heme binding. Nascent polypeptides of predetermined size were synthesized on ribosomes by translation of truncated mRNA molecules. Analysis with the use of sucrose gradient centrifugation and puromycin reaction revealed that the ribosome-bound N-terminal alpha-globin fragments of 140, 100, and 86 amino acid residues are capable of an efficient heme binding, whereas those of 75, 65, and 34 amino acid residues display a significantly weaker, or just nonspecific, affinity to heme. This indicates that the ribosome-bound nascent chain of 86 amino acid residues has already acquired a spatial structure that allows its interaction with the heme group or that heme attachment promotes the formation of the proper tertiary structure in the ribosome-bound nascent peptide. In any case the cotranslational folding of globin is suggested.