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Folding and escape of nascent proteins at ribosomal exit tunnel
Abstract
We investigate the interplay between post-translational folding and escape of two small single-domain
proteins at the ribosomal exit tunnel by using Langevin dynamics with coarse-grained models. It is
shown that at temperatures lower or near the temperature of the fastest folding, folding proceeds
concomitantly with the escape process, resulting in vectorial folding and enhancement of foldability
of nascent proteins. The concomitance between the two processes, however, deteriorates as temperature
increases. Our folding simulations as well as free energy calculation by using umbrella sampling
show that, at low temperatures, folding at the tunnel follows one or two specific pathways without
kinetic traps. It is shown that the escape time can be mapped to a one-dimensional diffusion model
with two different regimes for temperatures above and below the folding transition temperature.
Attractive interactions between amino acids and attractive sites on the tunnel wall lead to a free
energy barrier along the escape route of the protein. It is suggested that this barrier slows down the
escape process and consequently promotes correct folding of the released nascent protein.
Supplementary resource (1)
... Newly synthesized proteins are released from a ribosome through the exit tunnel. The latter impacts co-translational folding [1][2][3] of the nascent polypeptides as well as the post-translational escape [4][5][6][7] of nascent proteins. Despite the complex shape of the ribosome tunnel and its diverse interactions with nascent chains, recent simulation studies have shown that the escape process of nascent proteins is akin to simple diffusion given by a one-dimensional diffusion model [5][6][7]. ...
... The motions of nascent chains were simulated by using a molecular dynamics method based on the Langevin equation of motion and the Verlet algorithm [4]. All amino acids are assumed to have the same mass, m, and the same friction coefficient ζ . ...
... Interestingly, the escape time distribution in Eq. (1) can be well fitted to data from various simulations of protein escape in the Gō-like model [5][6][7]. It has been shown that the free energy of a protein at the ribosome tunnel is approximately linear along the escape coordinate [4,7], which justifies the linear form of U(x) in the diffusion model. It is quite clear that the shape and structure of the exit tunnel of H. marismortui and E. coli are very different [8,14]. ...
We study the escape process of nascent proteins at the ribosomal exit tunnel of bacterial Escherichia coli by using molecular dynamics simulations with coarse-grained and atomistic models. It is shown that the effects of hydrophobic and electrostatic interactions on the protein escape at the E. coli's tunnel are qualitatively similar to those obtained previously at the exit tunnel of archaeal Haloarcula marismortui, despite significant differences in the structures and interactions of the ribosome tunnels from the two organisms. Most proteins escape efficiently and their escape time distributions can be fitted to a simple diffusion model. Attractive interactions between nascent protein and the tunnel can significantly slow down the escape process, as shown for the CI2 protein. Interestingly, it is found that the median escape times of the considered proteins (excluding CI2) strongly correlate with the function \(N_h + 5.9 Q\) of the number of hydrophobic residues, \(N_h\), and the net charge, \(Q\), of a protein, with a correlation coefficient of 0.958 for the E. coli's tunnel. The latter result is in quantitative agreement with a previous result for the H. marismortui's tunnel.
... In contrast to co-translational folding, which depends on factors associated with the translation process, such as the vectorial nature of protein synthesis (6), the nonequilibrium effect of chain growth (16) and the translation rates (17)(18)(19)(20), posttranslational escape, and folding processes are more independent while they are still under the influence of the ribosome tunnel. Few studies have addressed these posttranslational processes (21)(22)(23)(24). ...
... In recent works (21)(22)(23), by using coarse-grained modeling and molecular dynamics simulations, we have shown the escape process is concomitant with the folding process, and these two processes promote each other leading to both an efficient escape and an improved folding efficiency (21). Our studies indicate that the protein escape at the ribosome tunnel is driven by the following: 1) an enthalpic force associated with the folding of the protein outside the tunnel, 2) an entropy gain as the chain emerges from the tunnel, and 3) the stochastic motion of a partially folded chain. ...
... In recent works (21)(22)(23), by using coarse-grained modeling and molecular dynamics simulations, we have shown the escape process is concomitant with the folding process, and these two processes promote each other leading to both an efficient escape and an improved folding efficiency (21). Our studies indicate that the protein escape at the ribosome tunnel is driven by the following: 1) an enthalpic force associated with the folding of the protein outside the tunnel, 2) an entropy gain as the chain emerges from the tunnel, and 3) the stochastic motion of a partially folded chain. ...
After translation, nascent proteins must escape the ribosomal exit tunnel to attain complete folding to their native states. This escape process also frees up the ribosome tunnel for a new translation job. In this study, we investigate the impacts of energetic interactions between the ribosomal exit tunnel and nascent proteins on the protein escape process by molecular dynamics simulations using partially coarse-grained models which incorporate hydrophobic and electrostatic interactions of the ribosome tunnel of H. marismortui with nascent proteins. We find that, in general, attractive interactions slow down the protein escape process whereas repulsive interactions speed it up. For the small globular proteins considered, the median escape time correlates with both the number of hydrophobic residues, Nh, and the net charge, Q, of a nascent protein. A correlation coefficient exceeding 0.96 is found for the relation between the median escape time and a combined quantity of Nh+5.9Q, suggesting that it is about 6 times more efficient to modulate the escape time by changing the total charge than the number of hydrophobic residues. The estimated median escape times are found in the sub-millisecond to millisecond range, indicating that the escape does not delay the ribosome recycling. For various types of the tunnel model, with and without hydrophobic and electrostatic interactions, the escape time distribution always follows a simple diffusion model, which describes the escape process as a downhill drift of a Brownian particle, suggesting that nascent proteins escape along barrier-less pathways at the ribosome tunnel.
... 14,15 All these effects are indicative of a highly conditional and coordinated folding of nascent protein at the ribosome, which is clearly different from refolding 16 of a denatured protein in aqueous solvent. There have been experiments [17][18][19] as well as simulations [20][21][22] that show that the folding efficiency of proteins is improved under biosynthesis conditions. It was also suggested that the impact of cotranslational folding is evolutionarily imprinted on the protein native states, as seen with an increased helix propensity 9 and a decreased compactness 23 of the chain near the C-terminus in the statistical analyses of protein structures from the protein data bank (PDB). ...
... A too slow escape would decrease the productivity of the ribosome, while a too fast escape would make the nascent protein vulnerable to aggregation, 24 as the partially folded protein may still have a large exposure of hydrophobic segments. In a recent study, 22 by using molecular dynamics (MD) simulations, we have shown that post-translational folding at the exit tunnel is concomitant with the escape process and that the tunnel induces a vectorial folding of the full-length protein. Such a folding has a greatly reduced number of pathways and leads to an improved folding efficiency. ...
... Such a folding has a greatly reduced number of pathways and leads to an improved folding efficiency. Interestingly, it has been also shown 22 that the escape time distribution of protein can be captured by a simple one-dimensional diffusion model of a particle in a linear potential field with an exact solution of the Smoluchowski equation. ...
How fast a post-translational nascent protein escapes from the ribosomal exit tunnel is relevant to its folding and protection against aggregation. Here, by using Langevin molecular dynamics, we show that non-local native interactions help decrease the escape time, and foldable proteins generally escape much faster than same-length, self-repulsive homopolymers at low temperatures. The escape process, however, is slowed down by the local interactions that stabilize the α-helices. The escape time is found to increase with both the tunnel length and the concentration of macromolecular crowders outside the tunnel. We show that a simple diffusion model described by the Smoluchowski equation with an effective linear potential can be used to map out the escape time distribution for various tunnel lengths and various crowder concentrations. The consistency between the simulation data and the diffusion model, however, is found only for the tunnel length smaller than a crossover length of 90 Å-110 Å, above which the escape time increases much faster with the tunnel length. It is suggested that the length of ribosomal exit tunnel has been selected by evolution to facilitate both the efficient folding and the efficient escape of single-domain proteins. We show that macromolecular crowders lead to an increase in the escape time, and attractive crowders are unfavorable for the folding of nascent polypeptide. Published by AIP Publishing. https://doi.
... 14,15 All these effects are indicative of a highly conditional and coordinated folding of nascent protein at the ribosome, which is clearly different from refolding 16 of a denatured protein in aqueous solvent. There have been experiments [17][18][19] as well as simulations [20][21][22] that show that the folding efficiency of proteins is improved under biosynthesis conditions. It was also suggested that the impact of cotranslational folding is evolutionarily imprinted on the protein native states, as seen with an increased helix propensity 9 and a decreased compactness 23 of the chain near the C-terminus in the statistical analyses of protein structures from the protein data bank (PDB). ...
... A too slow escape would decrease the productivity of the ribosome, while a too fast escape would make the nascent protein vulnerable to aggregation, 24 as the partially folded protein may still have a large exposure of hydrophobic segments. In a recent study, 22 by using molecular dynamics (MD) simulations, we have shown that post-translational folding at the exit tunnel is concomitant with the escape process and that the tunnel induces a vectorial folding of the full-length protein. Such a folding has a greatly reduced number of pathways and leads to an improved folding efficiency. ...
... Such a folding has a greatly reduced number of pathways and leads to an improved folding efficiency. Interestingly, it has been also shown 22 that the escape time distribution of protein can be captured by a simple one-dimensional diffusion model of a particle in a linear potential field with an exact solution of the Smoluchowski equation. ...
... The tunnel was modeled as a cylinder of varying radius, aligned along the z-axis, with z = 0 taken to be the position of the PTC. We included an additional 2Å for the van der Waals (vdW) radius of a virtual residue embedded in the tunnel wall [28]. The inner surface of the tunnel was treated as a weakly adsorbing rigid bounding wall with short-ranged interactions given by ...
... While various studies have also used MD simulations over the past few years to study the polypeptide escape and the impact of the tunnel properties (including [19,22,28,34]), our study is, to our knowledge, the first to comprehensively study how trapping occurs post-translationally in the exit tunnel at the scale of small sORF's. For larger proteins, Bui and Hoang compared various singledomain proteins of length comprised between 37 and 99 residues [20], and observed that these proteins can successfully escape from the tunnel, in agreement with our results for polypeptides of length ≥ 38. ...
The exit tunnel is the sub-compartment of the ribosome that contains the nascent polypeptide chain and as such, is involved in various vital functions, including regulation of translation and protein folding. As the geometry of the tunnel shows important differences across species, we focus on key geometrical features of eukaryote and prokaryote tunnels. We used a simple coarse-grained molecular dynamics model to study the role of the tunnel geometry in the post-translational escape of short proteins (sORF's), with lengths ranging from 6 to 56 amino acids. We found that the probability of escape for prokaryotes is one for all but the 12-mer chains. Moreover, proteins of this length have an extremely low escape probability in eukaryotes. A detailed examination of the associated single trajectories and energy profiles showed that these variations can be explained by the interplay between the protein configurational space and the confinement effects introduced by the constriction sites of the ribosome exit tunnel. For certain lengths, either one or both of the constriction sites can lead to the trapping of the protein in the "pocket" regions preceding these sites. As the distribution of existing sORF's indicate some bias in length that is consistent with our findings, we finally suggest that the constraints imposed by the tunnel geometry have impacted the evolution of sORF's.
... Furthermore, emergence from the tunnel may be delayed in case of fast and excessive folding. Varying secondary structures show distinct escape kinetics with α-helices that are usually faster than β-sheets [237,238]. Moreover, folded states of the nascent chain inside the tunnel contribute to the selective recruitment of cytosolic targeting factors to the ribosome. ...
Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease.
... Alternatively, the contacts may be turned on and off [288] during the simulation. Finally, the system may be enhanced with some repulsing walls, mimicking the chaperonin confinement [289],[D8] or ribosome [290][291][292][293], [D5]. ...
... In the context of knotted proteins, single molecule force spectroscopy techniques were shown to be particularly useful in controlling the topology of the unfolded state 46 . Similarly, both "in vivo" folding experiments 47 and appropriate simulation protocols [48][49][50] could be employed to test the possible role of cotranslational folding in determining the patterns detected for entangled motifs: double cysteine mutants would then be predicted to be more deleterious for the folding of C-terminal threads with respect to N-terminal threads. In all cases, it is essential to gather statistics over several different proteins before validating or rejecting our hypothesis; the signals that we reveal in this contribution are statistical in nature; therefore we do not expect all entangled loops to form late in the folding process nor all C-terminal threads to be cotranslationally disfavored. ...
Proteins must fold quickly to acquire their biologically functional three-dimensional native structures. Hence, these are mainly stabilized by local contacts, while intricate topologies such as knots are rare. Here, we reveal the existence of specific patterns adopted by protein sequences and structures to deal with backbone self-entanglement. A large scale analysis of the Protein Data Bank shows that loops significantly intertwined with another chain portion are typically closed by weakly bound amino acids. Why is this energetic frustration maintained? A possible picture is that entangled loops are formed only toward the end of the folding process to avoid kinetic traps. Consistently, these loops are more frequently found to be wrapped around a portion of the chain on their N-terminal side, the one translated earlier at the ribosome. Finally, these motifs are less abundant in natural native states than in simulated protein-like structures, yet they appear in 32% of proteins, which in some cases display an amazingly complex intertwining.
... Then, MD simulations can provide insight into molecular mechanism of complicated processes if experimental investigation is hampered. This can be illustrated by computational studies of nascent protein release from ribosomal tunnel, which proposed the mechanism of the polypeptide chain movement and the role of charged residues of ribosomal proteins [126][127][128]. Finally (and maybe most importantly), MD simulation is a powerful approach for comparative investigation of similar proteins' interaction and drug design. ...
Interaction of proteins with charged macromolecules is involved in many processes in cells. Firstly, there are many naturally occurred charged polymers such as DNA and RNA, polyphosphates, sulfated glycosaminoglycans, etc., as well as pronouncedly charged proteins such as histones or actin. Electrostatic interactions are also important for “generic” proteins, which are not generally considered as polyanions or polycations. Finally, protein behavior can be altered due to post-translational modifications such as phosphorylation, sulfation, and glycation, which change a local charge of the protein region. Herein we review molecular modeling for the investigation of such interactions, from model polyanions and polycations to unfolded proteins. We will show that electrostatic interactions are ubiquitous, and molecular dynamics simulations provide an outstanding opportunity to look inside binding and reveal the contribution of electrostatic interactions. Since a molecular dynamics simulation is only a model, we will comprehensively consider its relationship with the experimental data.
DNA toroids are compact torus-shaped bundles formed by one or multiple DNA molecules being condensed from the solution due to various condensing agents. It has been shown that the DNA toroidal bundles are twisted. However, the global conformations of DNA inside these bundles are still not well understood. In this study, we investigate this issue by solving different models for the toroidal bundles and performing replica-exchange molecular dynamics (REMD) simulations for self-attractive stiff polymers of various chain lengths. We find that a moderate degree of twisting is energetically favorable for toroidal bundles, yielding optimal configurations of lower energies than in other bundles corresponding to spool-like and constant radius of curvature arrangements. The REMD simulations show that the ground states of the stiff polymers are twisted toroidal bundles with the average twist degrees close to those predicted by the theoretical model. Constant-temperature simulations show that twisted toroidal bundles can be formed through successive processes of nucleation, growth, quick tightening and slow tightening of the toroid, with the two last processes facilitating the polymer threading through the toroid's hole. A relatively long chain of 512 beads has an increased dynamical difficulty to access the twisted bundle states due to the polymer's topological constraint. Interestingly, we also observed significantly twisted toroidal bundles with a sharp U-shaped region in the polymer conformation. It is suggested that this U-shaped region makes the formation of twisted bundles easier by effectively reducing the polymer length. This effect can be equivalent to having multiple chains in the toroid.
The ribosomal exit tunnel is the primary structure affecting the release of nascent proteins at the ribosome. The ribosomal exit tunnels from different species have elements of conservation and differentiation in structural and physico-chemical properties. In this study, by simulating the elongation and escape processes of nascent proteins at the ribosomal exit tunnels of four different organisms, we show that the escape process has conserved mechanisms across the domains of life. Specifically, it is found that the escape process of proteins follows the diffusion mechanism given by a simple diffusion model and the median escape time positively correlates with the number of hydrophobic residues and the net charge of a protein for all the exit tunnels considered. These properties hold for twelve distinct proteins considered in two slightly different and improved Gō-like models. It is also found that the differences in physico-chemical properties of the tunnels lead to quantitative differences in the protein escape times. In particular, the relatively strong hydrophobicity of the E. coli's tunnel and the unusually high number of negatively charged amino acids on the tunnel's surface of H. marismortui lead to substantially slower escapes of proteins at these tunnels than at those of S. cerevisisae and H. sapiens.
The exit tunnel is the sub-compartment of the ribosome that contains the nascent polypeptide chain and as such, is involved in various vital functions, including regulation of translation and protein folding. As the geometry of the tunnel shows important differences across species, we focus on key geometrical features of eukaryote and prokaryote tunnels. We used a simple coarse-grained molecular dynamics model to study the role of the tunnel geometry in the post-translational escape of short proteins (sORF's), with lengths ranging from 6 to 56 amino acids. We found that the probability of escape for prokaryotes is one for all but the 12-mer chains. Moreover, proteins of this length have an extremely low escape probability in eukaryotes. A detailed examination of the associated single trajectories and energy profiles showed that these variations can be explained by the interplay between the protein configurational space and the confinement effects introduced by the constriction sites of the ribosome exit tunnel. For certain lengths, either one or both of the constriction sites can lead to the trapping of the protein in the "pocket" regions preceding these sites. As the distribution of existing sORF's indicate some bias in length that is consistent with our findings, we finally suggest that the constraints imposed by the tunnel geometry have impacted the evolution of sORF's.
We study the post-translational escape of nascent proteins at the ribosomal exit tunnel with the consideration of a real shape atomistic tunnel based on the Protein Data Bank structure of the large ribosome subunit of archeon Haloarcula marismortui. Molecular dynamics simulations employing the Go-like model for the proteins show that at intermediate and high temperatures, including a presumable physiological temperature, the protein escape process at the atomistic tunnel is quantitatively similar to that at a cylinder tunnel of length L = 72 Å and diameter d = 16 Å. At low temperatures, the atomistic tunnel, however, yields an increased probability of protein trapping inside the tunnel, while the cylinder tunnel does not cause the trapping. All-β proteins tend to escape faster than all-α proteins, but this difference is blurred on increasing the protein’s chain length. A 29-residue zinc-finger domain is shown to be severely trapped inside the tunnel. Most of the single-domain proteins considered, however, can escape efficiently at the physiological temperature with the escape time distribution following the diffusion model proposed in our previous works. An extrapolation of the simulation data to a realistic value of the friction coefficient for amino acids indicates that the escape times of globular proteins are at the sub-millisecond scale. It is argued that this time scale is short enough for the smooth functioning of the ribosome by not allowing nascent proteins to jam the ribosome tunnel.
We study the post-translational escape of nascent proteins at the ribosomal exit tunnel by using the Go-like model for proteins and a real shape atomistic tunnel built on the protein data bank (PDB) structure of a ribosome of Haloarcula marismortui . The full translation and escape processes of the immunoglobulin binding B1 domain of protein G (GB1) at the tunnel were simulated by using Langevin dynamics. We show that at the simulation temperature corresponding to a physiological temperature, the escape time follows quite well the one-dimensional diffusion model proposed in our earlier works. The relationship between folding and escape obtained for the atomistic tunnel is similar to those obtained previously for the cylinder tunnel.
How fast a post-translational nascent protein escapes from the ribosomal exit tunnel is relevant to its folding and protection against aggregation. Here, by using Langevin molecular dynamics, we show that non-local native interactions help decrease the escape time, and foldable proteins generally escape much faster than same-length, self-repulsive homopolymers at low temperatures. The escape process, however, is slowed down by the local interactions that stabilize the α-helices. The escape time is found to increase with both the tunnel length and the concentration of macromolecular crowders outside the tunnel. We show that a simple diffusion model described by the Smoluchowski equation with an effective linear potential can be used to map out the escape time distribution for various tunnel lengths and various crowder concentrations. The consistency between the simulation data and the diffusion model, however, is found only for the tunnel length smaller than a crossover length of 90 Å–110 Å, above which the escape time increases much faster with the tunnel length. It is suggested that the length of ribosomal exit tunnel has been selected by evolution to facilitate both the efficient folding and the efficient escape of single-domain proteins. We show that macromolecular crowders lead to an increase in the escape time, and attractive crowders are unfavorable for the folding of nascent polypeptide.
It is, nowadays, possible to simulate biological processes in conditions that mimic the different cellular compartments. Several groups have performed these calculations using molecular models that vary in performance and accuracy. In many cases, the atomistic degrees of freedom have been eliminated, sacrificing both structural complexity and chemical specificity to be able to explore slow processes. In this review, we will discuss the insights gained from computer simulations on macromolecule diffusion, nuclear body formation, and processes involving the genetic material inside cell-mimicking spaces. We will also discuss the challenges to generate new models suitable for the simulations of biological processes on a cell scale and for cell-cycle-long times, including non-equilibrium events such as the co-translational folding, misfolding, and aggregation of proteins. A prominent role will be played by the wise choice of the structural simplifications and, simultaneously, of a relatively complex energetic description. These challenging tasks will rely on the integration of experimental and computational methods, achieved through the application of efficient algorithms.
Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.
Proper folding of deeply knotted proteins has a very low success rate even in
structure-based models which favor formation of the native contacts but have no
topological bias. By employing a structure-based model, we demonstrate that
cotranslational folding on a model ribosome may enhance the odds to form
trefoil knots for protein YibK without any need to introduce any non-native
contacts. The ribosome is represented by a repulsive wall that keeps elongating
the protein. On-ribosome folding proceeds through a a slipknot conformation. We
elucidate the mechanics and energetics of its formation. We show that the
knotting probability in on-ribosome folding is a function of temperature and
that there is an optimal temperature for the process. Our model often leads to
the establishment of the native contacts without formation of the knot.
The ability to assemble nano- and micro- sized colloidal components into highly ordered configurations is often cited as the basis for developing advanced materials. However, the dynamics of stochastic grain boundary formation and motion have not been quantified, which limits the ability to control and anneal polycrystallinity in colloidal based materials. Here we use optical microscopy, Brownian Dynamic simulations, and a new dynamic analysis to study grain boundary motion in quasi-2D colloidal bicrystals formed within inhomogeneous AC electric fields. We introduce "low-dimensional" models using reaction coordinates for condensation and global order that capture first passage times between critical configurations at each applied voltage. The resulting models reveal that equal sized domains at a maximum misorientation angle show relaxation dominated by friction limited grain boundary diffusion; and in contrast, asymmetrically sized domains with less misorientation display much faster grain boundary migration due to significant thermodynamic driving forces. By quantifying such dynamics vs. compression (voltage), kinetic bottlenecks associated with slow grain boundary relaxation are understood, which can be used to guide the temporal assembly of defect-free single domain colloidal crystals.
It has been observed for several proteins that slowing down the rate at which individual codons are translated can increase their probability of cotranslational protein folding, while speeding up codon translation can decrease it. Here we investigate whether or not this inverse relationship between translation speed and the cotranslational folding probability is a general phenomenon or if other scenarios are possible. We first derive chemical kinetic equations that relate individual codon translation rates to the probability that a domain will fold, populate an intermediate or misfold, and examine the cotranslational folding scenarios that are possible within these models. We find that speeding up codon translation through misfolding-prone segments can, in some cases, increase the folding probability of a domain immediately before the nascent protein is released from the ribosome and decrease its chances of misfolding. Thus, for some proteins fast-translating codons could be as important as slow-translating codons in coordinating cotranslational protein folding.
We have studied folding mechanisms of three small globular proteins: crambin, chymotrypsin inhibitor 2 (CI2), and the fyn Src Homology 3 domain (SH3) which are modeled by a Go-type Hamiltonian with the Lennard-Jones interactions. It is shown that folding is dominated by a well-defined sequencing of events as determined by establishment of particular contacts. The order of events depends primarily on the geometry of the native state. Variations in temperature, coupling strengths, and viscosity affect the sequencing scenarios to a rather small extent. The sequencing is strongly correlated with the distance of the contacting amino acids along the sequence. Thus α helices get established first. Crambin is found to behave like a single-route folder, whereas in CI2 and SH3 the folding trajectories are more diversified. The folding scenarios for CI2 and SH3 are consistent with experimental studies of their transition states. © 2000 American Institute of Physics.
Proteins are synthesized by the ribosome and generally must fold to become functionally active. Although it is commonly assumed
that the ribosome affects the folding process, this idea has been extremely difficult to demonstrate. We have developed an
experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers.
In T4 lysozyme, synthesized in a reconstituted in vitro translation system, the ribosome slows the formation of stable tertiary
interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold
and aggregate when free in solution, but they remain folding-competent near the ribosomal surface. Altogether, our results
suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient
de novo attainment of the native state.
Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein SufI can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding.
The ribosome is a large complex catalyst responsible for the synthesis of new proteins, an essential function for life. New proteins emerge from the ribosome through an exit tunnel as nascent polypeptide chains. Recent findings indicate that tunnel interactions with the nascent polypeptide chain might be relevant for the regulation of translation. However, the specific ribosomal structural features that mediate this process are unknown. Performing molecular dynamics simulations, we are studying the interactions between components of the ribosome exit tunnel and different chemical probes (specifically different amino acid side chains or monovalent inorganic ions). Our free-energy maps describe the physicochemical environment of the tunnel, revealing binding crevices and free-energy barriers for single amino acids and ions. Our simulations indicate that transport out of the tunnel could be different for diverse amino acid species. In addition, our results predict a notable protein–RNA interaction between a flexible 23S rRNA tetraloop (gate) and ribosomal protein L39 (latch) that could potentially obstruct the tunnel's exit. By relating our simulation data to earlier biochemical studies, we propose that ribosomal features at the exit of the tunnel can play a role in the regulation of nascent chain exit and ion flux. Moreover, our free-energy maps may provide a context for interpreting sequence-dependent nascent chain phenomenology.
• fragment-based search
• free-energy calculation
• molecular dynamics
• simulation
• translation
The folding-unfolding transition in small globular proteins can be described in general as a two-state equilibrium process, the transition being similar to a first-order phase transition. Many models of the folding process or the folding-unfolding transition have been proposed and studied in recent years. This review analyses the physical bases of these various models. Analyses from the statistical physical point of view are emphasized. Methods of prediction are discussed only when they have implications regarding the mechanism of the folding-unfolding transition.
While the classical view of protein folding kinetics relies on phenomenological models, and regards folding intermediates in a structural way, the new view emphasizes the ensemble nature of protein conformations. Although folding has sometimes been regarded as a linear sequence of events, the new view sees folding as parallel microscopic multi-pathway diffusion-like processes. While the classical view invoked pathways to solve the problem of searching for the needle in the haystack, the pathway idea was then seen as conflicting with Anfinsen's experiments showing that folding is pathway-independent (Levinthal's paradox). In contrast, the new view sees no inherent paradox because it eliminates the pathway idea: folding can funnel to a single stable state by multiple routes in conformational space. The general energy landscape picture provides a conceptual framework for understanding both two-state and multi-state folding kinetics. Better tests of these ideas will come when new experiments become available for measuring not just averages of structural observables, but also correlations among their fluctuations. At that point we hope to learn much more about the real shapes of protein folding landscapes.
The 62 kDa protein firefly luciferase folds very rapidly upon translation on eukaryotic ribosomes. In contrast, the chaperone-mediated refolding of chemically denatured luciferase occurs with significantly slower kinetics. Here we investigate the structural basis for this difference in folding kinetics. We find that an N-terminal domain of luciferase (residues 1-190) folds co-translationally, followed by rapid formation of native protein upon release of the full-length polypeptide from the ribosome. In contrast sequential domain formation is not observed during in vitro refolding. Discrete unfolding steps, corresponding to domain unfolding, are however observed when the native protein is exposed to increasing concentrations of denaturant. Thus, the co-translational folding reaction bears more similarities to the unfolding reaction than to refolding from denaturant. We propose that co-translational domain formation avoids intramolecular misfolding and may be critical in the folding of multidomain proteins.
Recent years have witnessed dramatic advances in our understanding of how newly translated proteins fold in the cell and the contribution of molecular chaperones to this process. Folding in the cell must be achieved in a highly crowded macromolecular environment, in which release of nonnative polypeptides into the cytosolic solution might lead to formation of potentially toxic aggregates. Here I review the cellular mechanisms that ensure efficient folding of newly translated proteins in vivo. De novo protein folding appears to occur in a protected environment created by a highly processive chaperone machinery that is directly coupled to translation. Genetic and biochemical analysis shows that several distinct chaperone systems, including Hsp70 and the cylindrical chaperonins, assist the folding of proteins upon translation in the cytosol of both prokaryotic and eukaryotic cells. The cellular chaperone machinery is specifically recruited to bind to ribosomes and protects nascent chains and folding intermediates from nonproductive interactions. In addition, initiation of folding during translation appears to be important for efficient folding of multidomain proteins.
We use Langevin dynamics simulations of a minimalist off-lattice model to study the translocation of a beta hairpin forming peptide through a tunnel that mimics the exit tunnel in a ribosome. We have computed the free energy of the peptide as a function of its position relative to the tunnel exit and also studied the properties of the conformational ensemble, when the peptide's position is restricted at different points along the tunnel. Confining the peptide within a sufficiently wide tunnel stabilizes the folded state. The protein then remains folded as it moves towards the tunnel exit. However, when the diameter D of the tunnel is below a certain critical value D(c), confinement destabilizes the folded state and forces the peptide to assume an extended configuration. In this case, as the peptide progresses towards the tunnel exit and eventually leaves the tunnel, it goes through a series of compact, misfolded conformations and eventually folds when it gets close to the exit. The critical tunnel diameter D(c) is comparable to the width of ribosomal tunnels. Our results suggest that co-translational folding is probably not universal, but rather a protein-specific phenomenon.
Patterns and forms adopted by nature are often the results of simple dynamical paradigms. Here we show that a growing self-interacting string attached to a tracking origin, modeled to resemble nascent polypeptides in vivo, develops helical structures which are more pronounced at the growing end. We also show that the dynamic growth ensemble shares several features of an equilibrium ensemble in which the growing end of the polymer is under an effective stretching force. A statistical analysis of native states of proteins shows that the signature of this nonequilibrium phenomenon has been fixed by evolution at the C terminus, the growing end of a nascent protein. These findings suggest how evolution may have built on the properties of a generic nonequilibrium growth process in favoring helical structures in nascent chains.
Several experiments have suggested that newly synthesized polypeptide chains can adopt helical structures deep within the ribosome exit tunnel. We hypothesize that confinement in the roughly cylindrical tunnel can entropically stabilize α-helices. The hypothesis is validated by using theory and simulations of coarse-grained off-lattice models. The model helix, which is unstable in the bulk, is stabilized in a cylindrical cavity provided the diameter (D) of the cylinder exceeds a critical value D*. When D < D* both the helical content and the helix–coil transition temperature (Tf) decrease abruptly. Surprisingly, we find that the stability of the α-helix depends on the number (N) of amino acid residues. Entropic stabilization, as measured by changes in Tf, increases nonlinearly as N increases. The simulation results are in quantitative agreement with a standard helix–coil theory that takes into account entropy cost of confining a polypeptide chain in a cylinder. The results of this work are in qualitative accord with most of the findings of a recent experiment in which N-dependent ribosome-induced helix stabilization of transmembrane sequences was measured by fluorescence resonance energy transfer.
• confinement effects
• cylindrical pores
• entropic stabilization
• folding
We consider six different secondary structures of proteins and construct two types of Go-type off-lattice models: with the steric constraints and without. The basic aminoacid-aminoacid potential is Lennard Jones for the native contacts and a soft repulsion for the non-native contacts. The interactions are chosen to make the target secondary structure be the native state of the system. We provide a thorough equilibrium and kinetic characterization of the sequences through the molecular dynamics simulations with the Langevin noise. Models with the steric constraints are found to be better folders and to be more stable, especially in the case of the $\beta$-structures. Phononic spectra for vibrations around the native states have low frequency gaps that correlate with the thermodynamic stability. Folding of the secondary structures proceeds through a well defined sequence of events. For instance, $\alpha$-helices fold from the ends first. The closer to the native state, the faster establishment of the contacts. Increasing the system size deteriorates the folding characteristics. We study the folding times as a function of viscous friction and find a regime of moderate friction with the linear dependence. We also consider folding when one end of a structure is pinned which imitates instantaneous conditions when a protein is being synthesized. We find that, under such circumstances, folding of helices is faster and of the $\beta$-sequences slower. Comment: REVTeX, 14 pages, EPS figures included, JCP in press
This text provides a uniform and consistent approach to diversified problems encountered in the study of dynamical processes in condensed phase molecular systems. Given the broad interdisciplinary aspect of this subject, the book focuses on three themes: coverage of needed background material, in-depth introduction of methodologies, and analysis of several key applications. The uniform approach and common language used in all discussions help to develop general understanding and insight on condensed phases chemical dynamics. The applications discussed are among the most fundamental processes that underlie physical, chemical and biological phenomena in complex systems. The first part of the book starts with a general review of basic mathematical and physical methods (Chapter 1) and a few introductory chapters on quantum dynamics (Chapter 2), interaction of radiation and matter (Chapter 3) and basic properties of solids (chapter 4) and liquids (Chapter 5). In the second part the text embarks on a broad coverage of the main methodological approaches. The central role of classical and quantum time correlation functions is emphasized in Chapter 6. The presentation of dynamical phenomena in complex systems as stochastic processes is discussed in Chapters 7 and 8. The basic theory of quantum relaxation phenomena is developed in Chapter 9, and carried on in Chapter 10 which introduces the density operator, its quantum evolution in Liouville space, and the concept of reduced equation of motions. The methodological part concludes with a discussion of linear response theory in Chapter 11, and of the spin-boson model in chapter 12. The third part of the book applies the methodologies introduced earlier to several fundamental processes that underlie much of the dynamical behaviour of condensed phase molecular systems. Vibrational relaxation and vibrational energy transfer (Chapter 13), Barrier crossing and diffusion controlled reactions (Chapter 14), solvation dynamics (Chapter 15), electron transfer in bulk solvents (Chapter 16) and at electrodes/electrolyte and metal/molecule/metal junctions (Chapter 17), and several processes pertaining to molecular spectroscopy in condensed phases (Chapter 18) are the main subjects discussed in this part.
In vivo, folding of many proteins occurs during their synthesis in the ribosomeand continues after they have escaped from the ribosomal exit tunnel. Inthis research, we investigate the confinement effects of the ribosome on thecotranslational folding of three proteins, of PDB codes 1PGA, 1CRN and 2RJX,by using a coarse-grained model and molecular dynamics simulation. The exittunnel is modeled as a hollow cylinder attached to a flat wall, whereas aGo-like model is adopted for the proteins. Our results show that theexit tunnel has a strong effect on the folding mechanism by setting an order bywhich the secondary and tertiary structures are formed. For protein 1PGA, thefolding follows two different folding routes. The presence of the tunnel alsoimproves the foldability of protein.
Experiments have demonstrated that changing the rate at which the ribosome translates a codon position in an mRNA molecule's open reading frame can alter the behavior of the newly synthesized protein. That is, codon translation rates can govern nascent proteome behavior. Here, we emphasize that this phenomenon is a manifestation of the non-equilibrium nature of co-translational processes, and, as such, there exist theoretical tools that offer a potential means to quantitatively predict the influence of codon translation rates on the broad spectrum of nascent protein behaviors including co-translational folding, aggregation and translocation. We provide a review of the experimental evidence for the impact that codon translation rates can have, followed by a discussion of theoretical methods that can describe this phenomenon. The development and application of these tools are likely to provide fundamental insights into protein maturation and homeostasis, codon usage bias in organisms, the origins of translation related diseases, and new rational design methods for biotechnology and biopharmaceutical applications.
We propose a method for identifying accurate reaction coordinates among a set of trial coordinates. The method applies to special cases where motion along the reaction coordinate follows a one-dimensional Smoluchowski equation. In these cases the reaction coordinate can predict its own short-time dynamical evolution, i.e., the dynamics projected from multiple dimensions onto the reaction coordinate depend only on the reaction coordinate itself. To test whether this property holds, we project an ensemble of short trajectory swarms onto trial coordinates and compare projections of individual swarms to projections of the ensemble of swarms. The comparison, quantified by the Kullback-Leibler divergence, is numerically performed for each isosurface of each trial coordinate. The ensemble of short dynamical trajectories is generated only once by sampling along an initial order parameter. The initial order parameter should separate the reactants and products with a free energy barrier, and distributions on isosurfaces of the initial parameter should be unimodal. The method is illustrated for three model free energy landscapes with anisotropic diffusion. Where exact coordinates can be obtained from Kramers-Langer-Berezhkovskii-Szabo theory, results from the new method agree with the exact results. We also examine characteristics of systems where the proposed method fails. We show how dynamical self-consistency is related (through the Chapman-Kolmogorov equation) to the earlier isocommittor criterion, which is based on longer paths.
The manner in which a newly synthesized chain of amino acids transforms itself into a perfectly folded protein depends both on the intrinsic properties of the amino-acid sequence and on multiple contributing influences from the crowded cellular milieu. Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases.
We show, by application to a simple protein folding model, how a stochastic Hamiltonian can be used to study a complex chemical reaction. This model is found to have many metastable states and the properties of these states are investigated. A simple generalization of transition-state theory is developed and used to estimate the folding time for the model. It is found to have a glass phase where the dynamics is very slow. The relevance of our results to protein folding and the general problem of complex chemical reactions is discussed.
The free energy difference between a model system and some reference system can easily be written as an ensemble average, but the conventional Monte Carlo methods of obtaining such averages are inadequate for the free-energy case. That is because the Boltzmann-weighted sampling distribution ordinarily used is extremely inefficient for the purpose. This paper describes the use of arbitrary sampling distributions chosen to facilitate such estimates. The methods have been tested successfully on the Lennard-Jones system over a wide range of temperature and density, including the gas-liquid coexistence region, and are found to be extremely powerful and economical.
Recent experimental results suggest that the native fold, or topology, plays a primary role in determining the structure of the transition state ensemble, at least for small, fast-folding proteins. To investigate the extent of the topological control of the folding process, we studied the folding of simplified models of five small globular proteins constructed using a G-like potential to retain the information about the native structures but drastically reduce the energetic frustration and energetic heterogeneity among residue-residue native interactions. By comparing the structure of the transition state ensemble (experimentally determined by Φ-values) and of the intermediates with those obtained using our models, we show that these energetically unfrustrated models can reproduce the global experimentally known features of the transition state ensembles and “en-route” intermediates, at least for the analyzed proteins. This result clearly indicates that, as long as the protein sequence is sufficiently minimally frustrated, topology plays a central role in determining the folding mechanism.
A particle which is caught in a potential hole and which, through the shuttling action of Brownian motion, can escape over a potential barrier yields a suitable model for elucidating the applicability of the transition state method for calculating the rate of chemical reactions.
The Weighted Histogram Analysis Method (WHAM), an extension of Ferrenberg and Swendsen's Multiple Histogram Technique, has been applied for the first time on complex biomolecular Hamiltonians. The method is presented here as an extension of the Umbrella Sampling method for free-energy and Potential of Mean Force calculations. This algorithm possesses the following advantages over methods that are currently employed: (1) It provides a built-in estimate of sampling errors thereby yielding objective estimates of the optimal location and length of additional simulations needed to achieve a desired level of precision; (2) it yields the “best” value of free energies by taking into account all the simulations so as to minimize the statistical errors; (3) in addition to optimizing the links between simulations, it also allows multiple overlaps of probability distributions for obtaining better estimates of the free-energy differences. By recasting the Ferrenberg–Swendsen Multiple Histogram equations in a form suitable for molecular mechanics type Hamiltonians, we have demonstrated the feasibility and robustness of this method by applying it to a test problem of the generation of the Potential of Mean Force profile of the pseudorotation phase angle of the sugar ring in deoxyadenosine. © 1992 by John Wiley & Sons, Inc.
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.
In living systems, polypeptide chains are synthesised on ribosomes, molecular machines composed of over 50 protein and nucleic acid molecules. As nascent chains emerge from the ribosomal exit tunnel and into the cellular environment, the majority must fold into specific structures in order to function. In this article we discuss recent approaches designed to reveal how such folding occurs and review our current knowledge of this complex self-assembly process.
The mechanisms for de novo protein folding differ significantly between bacteria and eukaryotes, as evidenced by the often observed poor yields of native eukaryotic proteins upon recombinant production in bacterial systems. Polypeptide synthesis rates are faster in bacteria than in eukaryotes, but the effects of general variations in translation rates on protein folding efficiency have remained largely unexplored. By employing Escherichia coli cells with mutant ribosomes whose translation speed can be modulated, we show here that reducing polypeptide elongation rates leads to enhanced folding of diverse proteins of eukaryotic origin. These results suggest that in eukaryotes, protein folding necessitates slow translation rates. In contrast, folding in bacteria appears to be uncoupled from protein synthesis, explaining our findings that a generalized reduction in translation speed does not adversely impact the folding of the endogenous bacterial proteome. Utilization of this strategy has allowed the production of a native eukaryotic multidomain protein that has been previously unattainable in bacterial systems and may constitute a general alternative to the production of aggregation-prone recombinant proteins.
Although tertiary folding of whole protein domains is prohibited by the cramped dimensions of the ribosomal tunnel, dynamic tertiary interactions may permit folding of small elementary units within the tunnel. To probe this possibility, we used a beta-hairpin and an alpha-helical hairpin from the cytosolic N terminus of a voltage-gated potassium channel and determined a probability of folding for each at defined locations inside and outside the tunnel. Minimalist tertiary structures can form near the exit port of the tunnel, a region that provides an entropic window for initial exploration of local peptide conformations. Tertiary subdomains of the nascent peptide fold sequentially, but not independently, during translation. These studies offer an approach for diagnosing the molecular basis for folding defects that lead to protein malfunction and provide insight into the role of the ribosome during early potassium channel biogenesis.
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 beta-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 beta-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 beta-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.
Diffusion in a spatially rough one-dimensional potential is treated by analysis of the mean first passage time. A general expression is found for the effective diffusion coefficient, which can become very small at low temperatures.
We present a new method for optimizing the analysis of data from multiple Monte Carlo computer simulations over wide ranges of parameter values. Explicit error estimates allow objective planning of the lengths of runs and the parameter values to be simulated. The method is applicable to simulations in lattice gauge theories, chemistry, and biology, as well as statistical mechanics.
The polypeptide chains that make up proteins have thousands of atoms and
hence millions of possible inter-atomic interactions. It might be supposed
that the resulting complexity would make prediction of protein structure and
protein-folding mechanisms nearly impossible. But the fundamental physics
underlying folding may be much simpler than this complexity would lead us
to expect: folding rates and mechanisms appear to be largely determined by
the topology of the native (folded) state, and new methods have shown great
promise in predicting protein-folding mechanisms and the three-dimensional
structures of proteins.
Four mutant strains from Saccharomyces cerevisiae were used to study ribosome structure and function. They included a strain carrying deletions of the two genes encoding ribosomal protein L24, a strain carrying a mutation spb2 in the gene for ribosomal protein L39, a strain carrying a deletion of the gene for L39, and a mutant lacking both L24 and L39. The mutant lacking only L24 showed just 25% of the normal polyphenylalanine-synthesizing activity followed by a decrease in P-site binding, suggesting the possibility that protein L24 is involved in the kinetics of translation. Each of the two L39 mutants displayed a 4-fold increase of their error frequencies over the wild type. This was accompanied by a substantial increase in A-site binding, typical of error-prone mutants. The absence of L39 also increased sensitivity to paromomycin, decreased the ribosomal subunit ratio, and caused a cold-sensitive phenotype. Mutant cells lacking both ribosomal proteins remained viable. Their ribosomes showed reduced initial rates caused by the absence of L24 but a normal extent of polyphenylalanine synthesis and a substantial in vivo reduction in the amount of 80S ribosomes compared to wild type. Moreover, this mutant displayed decreased translational accuracy, hypersensitivity to the antibiotic paromomycin, and a cold-sensitive phenotype, all caused mainly by the deletion of L39. Protein L39 is the first protein of the 60S ribosomal subunit implicated in translational accuracy.
Translation of SecM stalls unless its N-terminal part is "pulled" by the protein export machinery. Here we show that the sequence motif FXXXXWIXXXXGIRAGP that includes a specific arrest point (Pro) causes elongation arrest within the ribosome. Mutations that bypass the elongation arrest were isolated in 23S rRNA and L22 r protein. Such suppressor mutations occurred at a few specific residues of these components, which all face the narrowest constriction of the ribosomal exit tunnel. Thus, we suggest that this region of the exit tunnel interacts with nascent translation products and functions as a discriminating gate.
Our studies of SecM (secretion monitor) in E. coli have revealed that some amino acid sequences can interact with ribosomal interior components, particularly with gate components of the exit tunnel, thereby interfering with their own translation elongation. Such translation arrest can be regulated by interaction of the N-terminal portion of the nascent polypeptide with other cellular components outside the ribosome. These properties of nascent proteins can in turn provide regulatory mechanisms by which the expression of genetic information at different levels is regulated.
The capsid protein of Semliki Forest virus constitutes the N-terminal part of a large viral polyprotein. It consists of an unstructured basic segment (residues 1-118) and a 149 residue serine protease module (SFVP, residues 119-267) comprised of two beta-barrel domains. Previous in vivo and in vitro translation experiments have demonstrated that SFVP folds co-translationally during synthesis of the viral polyprotein and rapidly cleaves itself off the nascent chain. To test whether fast co-translation folding of SFVP is an intrinsic property of the polypeptide chain or whether folding is accelerated by cellular components, we investigated spontaneous folding of recombinant SFVP in vitro. The results show that the majority of unfolded SFVP molecules fold faster than any previously studied two-domain protein (tau=50 ms), and that folding of the N-terminal domain precedes structure formation of the C-terminal domain. This shows that co-translational folding of SFVP does not require additional cellular components and suggests that rapid folding is the result of molecular evolution towards efficient virus biogenesis.
Helicity of membrane proteins can be manifested inside the ribosome tunnel, but the determinants of compact structure formation inside the tunnel are largely unexplored. Using an extended nascent peptide as a molecular tape measure of the ribosomal tunnel, we have previously demonstrated helix formation inside the tunnel. Here, we introduce a series of consecutive polyalanines into different regions of the tape measure to monitor the formation of compact structure in the nascent peptide. We find that the formation of compact structure of the polyalanine sequence depends on its location. Calculation of free energies for the equilibria between folded and unfolded nascent peptides in different regions of the tunnel shows that there are zones of secondary structure formation inside the ribosomal exit tunnel. These zones may have an active role in nascent-chain compaction.
The geometry of the polypeptide exit tunnel has been determined using the crystal structure of the large ribosomal subunit from Haloarcula marismortui. The tunnel is a component of a much larger, interconnected system of channels accessible to solvent that permeates the subunit and is connected to the exterior at many points. Since water and other small molecules can diffuse into and out of the tunnel along many different trajectories, the large subunit cannot be part of the seal that keeps ions from passing through the ribosome-translocon complex. The structure referred to as the tunnel is the only passage in the solvent channel system that is both large enough to accommodate nascent peptides, and that traverses the particle. For objects of that size, it is effectively an unbranched tube connecting the peptidyl transferase center of the large subunit and the site where nascent peptides emerge. At no point is the tunnel big enough to accommodate folded polypeptides larger than alpha-helices.
The viscosity dependence of the folding rates for four sequences (the native state of three sequences is a beta-sheet, while the fourth forms an alpha-helix) is calculated for off-lattice models of proteins. Assuming that the dynamics is given by the Langevin equation we show that the folding rates increase linearly at low viscosities \eta, decrease as 1/\eta at large \eta and have a maximum at intermediate values. The Kramers theory of barrier crossing provides a quantitative fit of the numerical results. By mapping the simulation results to real proteins we estimate that for optimized sequences the time scale for forming a four turn \alpha-helix topology is about 500 nanoseconds, whereas the time scale for forming a beta-sheet topology is about 10 microseconds. Comment: 14 pages, Latex, 3 figures. One figure is also available at http://www.glue.umd.edu/~klimov/seq_I_H.html, to be published in Physical Review Letters
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