Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.     

Cotranslational Folding Increases GFP Folding Yield

Protein sequences evolved to fold in cells, including cotranslational folding of nascent polypeptide chains during their synthesis by the ribosome. The vectorial (N- to C-terminal) nature of cotranslational folding constrains the conformations of the nascent polypeptide chain in a manner not experienced by full-length chains diluted out of denaturant. We are still discovering to what extent these constraints affect later, posttranslational folding events. Here we directly address whether conformational constraints imposed by cotranslational folding affect the partitioning between productive folding to the native structure versus aggregation. We isolated polyribosomes from Escherichia coli cells expressing GFP, analyzed the nascent chain length distribution to determine the number of nascent chains that were long enough to fold to the native fluorescent structure, and calculated the folding yield for these nascent chains upon ribosome release versus the folding yield of an equivalent concentration of full-length, chemically denatured GFP polypeptide chains. We find that the yield of native fluorescent GFP is dramatically higher upon ribosome release of nascent chains versus dilution of full-length chains from denaturant. For kinetically trapped native structures such as GFP, folding correctly the first time, immediately after release from the ribosome, can lead to lifelong population of the native structure, as opposed to aggregation.     

Co-translational binding of GroEL to nascent polypeptides is followed by post-translational encapsulation by GroES to mediate protein folding

The eubacterial chaperonins GroEL and GroES are essential chaperones and primarily assist protein folding in the cell. Although the molecular mechanism of the GroEL system has been examined previously, the mechanism by which GroEL and GroES assist folding of nascent polypeptides during translation is still poorly understood. We previously demonstrated a co-translational involvement of the Escherichia coli GroEL in folding of newly synthesized polypeptides using a reconstituted cell-free translation system (Ying, B. W., Taguchi, H., Kondo, M., and Ueda, T. (2005) J. Biol. Chem. 280, 12035-12040). Employing the same system here, we further characterized the mechanism by which GroEL assists folding of translated proteins via encapsulation into the GroEL-GroES cavity. The stable co-translational association between GroEL and the newly synthesized polypeptide is dependent on the length of the nascent chain. Furthermore, GroES is capable of interacting with the GroEL-nascent peptide-ribosome complex, and experiments using a single-ring variant of GroEL clearly indicate that GroES association occurs only at the trans-ring, not the cis-ring, of GroEL. GroEL holds the nascent chain on the ribosome in a polypeptide length-dependent manner and post-translationally encapsulates the polypeptide using the GroES cap to accomplish the chaperonin-mediated folding process.     

A model study of protein nascent chain and cotranslational folding using hydrophobic-polar residues

To study protein nascent chain folding during biosynthesis, we investigate the folding behavior of models of hydrophobic and polar (HP) chains at growing length using both two-dimensional square lattice model and an optimized three-dimensional 4-state discrete off-lattice model. After enumerating all possible sequences and conformations of HP heteropolymers up to length N = 18 and N = 15 in two and three-dimensional space, respectively, we examine changes in adopted structure, stability, and tolerance to single point mutation as the nascent chain grows. In both models, we find that stable model proteins have fewer folded nascent chains during growth, and often will only fold after reaching full length. For the few occasions where partial chains of stable proteins fold, these partial conformations on average are very similar to the corresponding parts of the final conformations at full length. Conversely, we find that sequences with fewer stable nascent chains and sequences with native-like folded nascent chains are more stable. In addition, these stable sequences in general can have many more point mutations and still fold into the same conformation as the wild type sequence. Our results suggest that stable proteins are less likely to be trapped in metastable conformations during biosynthesis, and are more resistant to point-mutations. Our results also imply that less stable proteins will require the assistance of chaperone and other factors during nascent chain folding. Taken together with other reported studies, it seems that cotranslational folding may not be a general mechanism of in vivo protein folding for small proteins, and in vitro folding studies are still relevant for understanding how proteins fold biologically.     

The alpha and beta subunit of the nascent polypeptide-associated complex have distinct functions

Nascent polypeptide-associated complex (NAC) is probably the first cytosolic protein to contact nascent polypeptide chains emerging from ribosomes. In this way NAC prevents inappropriate interactions with other factors. Eventually other factors involved in targeting and folding, like the Signal Recognition Particle or cytosolic chaperones, must gain access to the nascent chain. All NAC preparations to date consist of two copurifying polypeptides. Here we rigorously show that these two polypeptides, termed alpha- and betaNAC, form a very stable complex in vivo and in vitro and that a functional complex can be reconstituted from the individual subunits. A dissection of the contributions of the individual subunits to NACs function revealed that both subunits are in direct contact with nascent polypeptide chains on the ribosome and that both contribute to the prevention of inappropriate interactions. However, betaNAC alone directly binds to the ribosome and is sufficient to prevent ribosome binding to the endoplasmic reticulum membrane.     

Role of the ribosome in protein folding

In all organisms, the ribosome synthesizes and folds full length polypeptide chains into active three-dimensional conformations. The nascent protein goes through two major interactions, first with the ribosome which synthesizes the polypeptide chain and holds it for a considerable length of time, and then with the chaperones. Some of the chaperones are found in solution as well as associated to the ribosome. A number of in vitro and in vivo experiments revealed that the nascent protein folds through specific interactions of some amino acids with the nucleotides in the peptidyl transferase center (PTC) in the large ribosomal subunit. The mechanism of this folding differs from self-folding. In this article, we highlight the folding of nascent proteins on the ribosome and the influence of chaperones etc. on protein folding.     

Cotranslational folding promotes beta-helix formation and avoids aggregation in vivo

Newly synthesized proteins must form their native structures in the crowded environment of the cell, while avoiding non-native conformations that can lead to aggregation. Yet, remarkably little is known about the progressive folding of polypeptide chains during chain synthesis by the ribosome or of the influence of this folding environment on productive folding in vivo. P22 tailspike is a homotrimeric protein that is prone to aggregation via misfolding of its central β-helix domain in vitro. We have produced stalled ribosome:tailspike nascent chain complexes of four fixed lengths in vivo, in order to assess cotranslational folding of newly synthesized tailspike chains as a function of chain length. Partially synthesized, ribosome-bound nascent tailspike chains populate stable conformations with some native-state structural features even prior to the appearance of the entire β-helix domain, regardless of the presence of the chaperone trigger factor, yet these conformations are distinct from the conformations of released, refolded tailspike truncations. These results suggest that organization of the aggregation-prone β-helix domain occurs cotranslationally, prior to chain release, to a conformation that is distinct from the accessible energy minimum conformation for the truncated free chain in solution.     

Protein folding in vitro and in the cellular environment

The main concepts concerning protein folding have been developed from in vitro refolding studies. They state that the folding of a polypeptide chain is a spontaneous process depending only on the amino-acid sequence in a given environment. It is thermodynamically controlled and driven by the hydrophobic effect. Consequently, it has been accepted that the in vitro refolding process is a valuable model to understand the mechanisms involved during the folding of a nascent polypeptide chain in the cell. Although it does not invalidate the main rules deduced from the in vitro studies, the discovery of molecular chaperones has led to a re-evaluation of this last point. Indeed, in cells molecular chaperones are able to mediate the folding of polypeptide chains and the assembly of subunits in oligomeric proteins. The possible mechanisms by which these folding helpers act are discussed in the light of the data available in the literature. The folding process is assisted in the cell in different ways, preventing premature folding of the polypeptide chain and suppressing the incorrectly folded species and aggregates. Molecular chaperones bind to incompletely folded proteins in a conformation which suggests that the latter are in the "molten globule" state. However, very little is known about the recognition process.     

The co-translational folding and interactions of nascent protein chains: a new approach using fluorescence resonance energy transfer

During protein biosynthesis, a nascent protein is exposed to multiple environments and proteins both inside and outside the ribosome that influence nascent chain folding and trafficking. Fluorescence resonance energy transfer between two dyes incorporated into a single nascent chain using aminoacyl-tRNA analogs can directly and selectively monitor changes in nascent chain conformation. This approach recently revealed the existence and functional ramifications of ribosome-mediated folding of nascent membrane proteins inside the ribosome and can be extended to characterize the effects of chaperones and other proteins and ligands on nascent protein folding, interactions, assembly, and avoidance of misfolding and degradation.     

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process^1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon⁶. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site^7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes⁷. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains^10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.     

Full Reconstruction of a Vectorial Protein Folding Pathway by Atomic Force Microscopy and Molecular Dynamics Simulations

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and, under severe conformational constraints, is dictated by its one-dimensional geometry. How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.     

An in vitro peptide folding model suggests the presence of the molten globule state during nascent peptide folding

Although molten globules have been widely accepted as a general intermediate in protein folding, there is no clear evidence to show their presence during nascent peptide folding. This paper concentrates on whether the molten globule state occurs, and if it does, when does it form during nascent peptide folding, by comparing the changes in conformation during peptide chain extension of staphylococcal nuclease R. The results show that a large N-terminal fragment of staphylococcal nuclease, SNR121, which already contains more than 80% amino acid sequence of the nuclease, is found to fulfill all the criteria for the molten globule state, suggesting that the molten globule should occur at a later stage of peptide elongation. At this stage the hydrophobic collapse of the polypeptide chain occurs driven by the hydrophobic force, which leads to the formation of a solvent-accessible non-polar core, characterized by the high ANS-binding fluorescence. The nascent peptide folding of the nuclease is a hierarchical process that at the very least includes the following steps: secondary structure accumulation, pre-molten globule state, molten globule state, post-molten globule state and finally the native state. Constant conformation adjustment is necessary for correct folding and active expression of the protein.     

Roles of molecular chaperones in cytoplasmic protein folding

Newly synthesized polypeptide chains must fold and assemble into unique three-dimensional structures in order to become functionally active. In many cases productive folding depends on assistance from molecular chaperones, which act in preventing off-pathway reactions during folding that lead to aggregation. The inherent tendency of incompletely folded polypeptide chains to aggregate is thought to be strongly enhanced$L in vivo *I$Lby the high macromolecular concentration of the cellular solution, resulting in crowding effects, and by the close proximity of nascent polypeptide chains during synthesis on polyribosomes. The major classes of chaperones acting in cytoplasmic protein folding are the Hsp70s and the chaperonins. Hsp70 chaperones shield the hydrophobic regions of nascent and incompletely folded chains, whereas the chaperonins provide a sequestered environment in which folding can proceed unimpaired by intermolecular interactions between non-native polypeptides. These two principles of chaperone action can function in a coordinated manner to ensure the efficient folding of a subset of cytoplasmic proteins.     

Folding at the birth of the nascent chain: coordinating translation with co-translational folding

In the living cells, the folding of many proteins is largely believed to begin co-translationally, during their biosynthesis at the ribosomes. In the ribosomal tunnel, the nascent peptide may establish local interactions and stabilize α-helical structures. Long-range contacts are more likely outside the ribosomes after release of larger segments of the nascent chain. Examples suggest that domains can attain native-like structure on the ribosome with and without population of folding intermediates. The co-translational folding is limited by the speed of the gradual extrusion of the nascent peptide which imposes conformational restraints on its folding landscape. Recent experimental and in silico modeling studies indicate that translation kinetics fine-tunes co-translational folding by providing a time delay for sequential folding of distinct portions of the nascent chain.     

The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling

As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit and emerges at the solvent side where protein folding occurs. Despite the universality and conservation of dimensions of the ribosomal tunnel, a functional role for the ribosomal tunnel is only beginning to emerge: Rather than a passive conduit for the nascent chain, accumulating evidence indicates that the tunnel plays a more active role. In this article, we discuss recent structural insights into the role of the tunnel environment, and its implications for protein folding, co-translational targeting and translation regulation.     

N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin

For proteins that traverse the secretory pathway, folding commences cotranslationally upon translocation into the endoplasmic reticulum. In this study, we have comprehensively analyzed the earliest maturation steps of the model glycoprotein influenza hemagglutinin (HA). These steps include cleavage of the signal sequence, glycosylation, binding by the chaperones calnexin and calreticulin, and the oxidoreductase ERp57, and oxidation. Our results show that the molecular choreography of the nascent HA chain is largely directed by multiple glycans that are strategically placed to elicit the binding of lectin chaperones. These chaperones are recruited to specific nascent chain locations to regulate and facilitate glycoprotein folding, thereby suggesting that the positioning of N-linked glycans in critical regions has evolved to optimize the folding process in the cell.     

Generation of ribosome nascent chain complexes for structural and functional studies

Biochemical and structural studies of co-translational folding, targeting and translocation depend on an efficient methodology to prepare ribosome nascent chain complexes (RNCs). Here we present our approach for the generation of homogenous and stable RNCs involving in vitro translation and affinity purification. Fusing the SecM arrest sequence, which tightly interacts with the ribosomal tunnel, to the nascent polypeptide chain significantly enhanced the stability of the RNCs. We have been able to increase the yield of the affinity purification step by engineering a tag with higher affinity. The RNCs generated with this approach have been successfully used to obtain 3D cryo-electron microscopic reconstructions of complexes with the signal recognition particle and the translocon. The established procedure is highly efficient and if scaled up could yield milligram amounts of RNCs sufficient for crystallization experiments.