The folding and unfolding behavior of ribonuclease H on the ribosome.

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome in vitro We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.

Folding pathway of an Ig domain is conserved on and off the ribosome.

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-ß Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.

Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins.

A large range of debilitating medical conditions is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains. Seventy-five per cent of the eukaryotic proteome consists of multidomain proteins, yet it is not understood how interdomain misfolding is avoided. It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding. Here we use single-molecule Förster resonance energy transfer to detect and quantify rare misfolding events in tandem immunoglobulin domains from the I band of titin under native conditions. About 5.5 per cent of molecules with identical domains misfold during refolding in vitro and form an unexpectedly stable state with an unfolding half-time of several days. Tandem arrays of immunoglobulin-like domains in humans show significantly lower sequence identity between neighbouring domains than between non-adjacent domains. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent. We observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain-swapping mechanism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.

Experimental evidence for a frustrated energy landscape in a three-helix-bundle protein family.

Energy landscape theory is a powerful tool for understanding the structure and dynamics of complex molecular systems, in particular biological macromolecules. The primary sequence of a protein defines its free-energy landscape and thus determines the folding pathway and the rate constants of folding and unfolding, as well as the protein's native structure. Theory has shown that roughness in the energy landscape will lead to slower folding, but derivation of detailed experimental descriptions of this landscape is challenging. Simple folding models show that folding is significantly influenced by chain entropy; proteins in which the contacts are local fold quickly, owing to the low entropy cost of forming stabilizing, native contacts during folding. For some protein families, stability is also a determinant of folding rate constants. Where these simple metrics fail to predict folding behaviour, it is probable that there are features in the energy landscape that are unusual. Such general observations cannot explain the folding behaviour of the R15, R16 and R17 domains of alpha-spectrin. R15 folds approximately 3,000 times faster than its homologues, although they have similar structures, stabilities and, as far as can be determined, transition-state stabilities. Here we show that landscape roughness (internal friction) is responsible for the slower folding and unfolding of R16 and R17. We use chimaeric domains to demonstrate that this internal friction is a property of the core, and suggest that frustration in the landscape of the slow-folding spectrin domains may be due to misdocking of the long helices during folding. Theoretical studies have suggested that rugged landscapes will result in slower folding; here we show experimentally that such a phenomenon directly influences the folding kinetics of a 'normal' protein, that is, one with a significant energy barrier that folds on a relatively slow, millisecond-second, timescale.

Staphylococcal biofilm-forming protein has a contiguous rod-like structure.

Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein (S. epidermidis) promote biofilm formation through their "B" regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn(2+)-mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E-G5 and G5-E-G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn(2+) does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the "zinc zipper." Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.

Folding and binding of an intrinsically disordered protein: fast, but not 'diffusion-limited'.

Coupled folding and binding of intrinsically disordered proteins (IDPs) is prevalent in biology. As the first step toward understanding the mechanism of binding, it is important to know if a reaction is 'diffusion-limited' as, if this speed limit is reached, the association must proceed through an induced fit mechanism. Here, we use a model system where the 'BH3 region' of PUMA, an IDP, forms a single, contiguous α-helix upon binding the folded protein Mcl-1. Using stopped-flow techniques, we systematically compare the rate constant for association (k(+)) under a number of solvent conditions and temperatures. We show that our system is not 'diffusion-limited', despite having a k(+) in the often-quoted 'diffusion-limited' regime (10(5)-10(6) M(-1) s(-1) at high ionic strength) and displaying an inverse dependence on solvent viscosity. These standard tests, developed for folded protein-protein interactions, are not appropriate for reactions where one protein is disordered.

Distinguishing specific and nonspecific interdomain interactions in multidomain proteins.

Multidomain proteins account for over two-thirds of the eukaryotic genome. Although there have been extensive studies into the biophysical properties of isolated domains, few have investigated how the domains interact. Spectrin is a well-characterized multidomain protein with domains linked in tandem array by contiguous helices. Several of these domains have been shown to be stabilized by their neighbors. Until now, this stabilization has been attributed to specific interactions between the natural neighbors, however we have recently observed that nonnatural neighboring domains can also induce a significant amount of stabilization. Here we investigate this nonnative stabilizing effect. We created spectrin-titin domain pairs of both spectrin R16 and R17 with a single titin I27 domain at either the N- or the C-terminus and found that spectrin domains are significantly stabilized, through slowed unfolding, by nonnative interactions at the C-terminus only. Of particular importance, we show that specific interactions between natural folded neighbors at either terminus confer even greater stability by additionally increasing the folding rate constants. We demonstrate that it is possible to distinguish between natural stabilizing interactions and nonspecific stabilizing effects through examination of the kinetics of well chosen mutant proteins. This work adds to the complexity of studying multidomain proteins.

Investigating the Effect of Chain Connectivity on the Folding of a Beta-Sheet Protein On and Off the Ribosome.

Determining the relationship between protein folding pathways on and off the ribosome remains an important area of investigation in biology. Studies on isolated domains have shown that alteration of the separation of residues in a polypeptide chain, while maintaining their spatial contacts, may affect protein stability and folding pathway. Due to the vectorial emergence of the polypeptide chain from the ribosome, chain connectivity may have an important influence upon cotranslational folding. Using MATH, an all ß-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome. From Φ-value analysis, we show that the most structured region of the transition state for folding in MATH includes the N and C terminal strands, which are located adjacent to each other in the structure. However, arrest peptide force-profile assays show that wild-type MATH is able to fold cotranslationally, while some C-terminal residues remain sequestered in the ribosome, even when destabilized by 2-3 kcal mol-1. We show that, while this pattern of Φ-values is retained in two circular permutants in our studies of the isolated domains, one of these permutants can fold only when fully emerged from the ribosome. We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both ß-sheets, rather than by the location of the terminal strands in the ribosome tunnel.

Chromatin accessibility dynamics across C. elegans development and ageing.

An essential step for understanding the transcriptional circuits that control development and physiology is the global identification and characterization of regulatory elements. Here, we present the first map of regulatory elements across the development and ageing of an animal, identifying 42,245 elements accessible in at least one Caenorhabditis elegans stage. Based on nuclear transcription profiles, we define 15,714 protein-coding promoters and 19,231 putative enhancers, and find that both types of element can drive orientation-independent transcription. Additionally, more than 1000 promoters produce transcripts antisense to protein coding genes, suggesting involvement in a widespread regulatory mechanism. We find that the accessibility of most elements changes during development and/or ageing and that patterns of accessibility change are linked to specific developmental or physiological processes. The map and characterization of regulatory elements across C. elegans life provides a platform for understanding how transcription controls development and ageing.

Cotranslational folding of spectrin domains via partially structured states.

How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes.

Cooperative folding in a multi-domain protein.

Most protein domains are found in multi-domain proteins, yet most studies of protein folding have concentrated on small, single-domain proteins or on isolated domains from larger proteins. Spectrin domains are small (106 amino acid residues), independently folding domains consisting of three long alpha-helices. They are found in multi-domain proteins with a number of spectrin domains in tandem array. Structural studies have shown that in these arrays the last helix of one domain forms a continuous helix with the first helix of the following domain. It has been demonstrated that a number of spectrin domains are stabilised by their neighbours. Here we investigate the molecular basis for cooperativity between adjacent spectrin domains 16 and 17 from chicken brain alpha-spectrin (R16 and R17). We show that whereas the proteins unfold as a single cooperative unit at 25 degrees C, cooperativity is lost at higher temperatures and in the presence of stabilising salts. Mutations in the linker region also cause the cooperativity to be lost. However, the cooperativity does not rely on specific interactions in the linker region alone. Most mutations in the R17 domain cause a decrease in cooperativity, whereas proteins with mutations in the R16 domain still fold cooperatively. We propose a mechanism for this behaviour.

Mechanical unfolding of TNfn3: the unfolding pathway of a fnIII domain probed by protein engineering, AFM and MD simulation.

Protein engineering Phi-value analysis combined with single molecule atomic force microscopy (AFM) was used to probe the molecular basis for the mechanical stability of TNfn3, the third fibronectin type III domain from human tenascin. This approach has been adopted previously to solve the forced unfolding pathway of a titin immunoglobulin domain, TI I27. TNfn3 and TI I27 are members of different protein superfamilies and have no sequence identity but they have the same beta-sandwich structure consisting of two antiparallel beta-sheets. TNfn3, however, unfolds at significantly lower forces than TI I27. We compare the response of these proteins to mechanical force. Mutational analysis shows that, as is the case with TI I27, TNfn3 unfolds via a force-stabilised intermediate. The key event in forced unfolding in TI I27 is largely the breaking of hydrogen bonds and hydrophobic interactions between the A' and G-strands. The mechanical Phi-value analysis and molecular dynamics simulations reported here reveal that significantly more of the TNfn3 molecule contributes to its resistance to force. Both AFM experimental data and molecular dynamics simulations suggest that the rate-limiting step of TNfn3 forced unfolding reflects a transition from the extended early intermediate to an aligned intermediate state. As well as losses of interactions of the A and G-strands and associated loops there are rearrangements throughout the core. As was the case for TI I27, the forced unfolding pathway of TNfn3 is different from that observed in denaturant studies in the absence of force.

Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments.

Sequence conservation in Ig-like domains: the role of highly conserved proline residues in the fibronectin type III superfamily.

The role of conserved proline residues in fibronectin type III (fnIII) domains is investigated. Surprisingly, none of the standard set of explanations for residue conservation applies. The proline residues are not apparently conserved for function, or stability, or to nucleate folding, or to promote stabilising interactions across domain boundaries. However, when the most highly conserved proline residues are mutated to alanine there is an increase in the rate of aggregation of a fnIII double-module construct. The results suggest that proline residues may be conserved at domain-domain boundaries in fnIII domains to prevent aggregation in multi-modular proteins.

Thermodynamic characterisation of two transition states along parallel protein folding pathways.

Recent work has shown that a beta-sandwich domain from the human muscle protein titin (TI I27) unfolds via more than one pathway, providing experimental evidence for a long-standing theoretical prediction in protein folding. Here we present a thermodynamic analysis of two transition states along different folding pathways for this protein. The unusual upwards curvature previously observed in the denaturant-dependent unfolding kinetics is increased at both high and low temperatures, indicating that the high denaturant pathway is becoming more accessible. The transition states in each pathway are structurally distinct and have very different heat capacities. Interestingly the nucleation-condensation pathway is dominant at all physiologically relevant temperatures, supporting the suggestion that pathways with diffuse rather than localised transition states have been selected for by evolution to prevent misfolding.

Titin; a multidomain protein that behaves as the sum of its parts.

Titin is a giant, multidomain muscle protein forming a major component of the sarcomere in vertebrate striated muscle. As for many other multidomain proteins, the properties of titin are often studied by characterisation of the constituent domains in isolation. This raises the question of to what extent the properties of the isolated domains are representative of the domains in the wild-type protein. We address this question for the I-band region of titin, which is of particular biological interest due to its role in muscle elasticity, by determining the properties of five immunoglobulin domains from the I-band in three different contexts; firstly as isolated domains with the boundaries defined conservatively, secondly, with a two amino acid extension at both the N and C terminus and thirdly as part of multidomain constructs. We show that adjacent domains in the titin I-band have very different kinetic properties which, in general, undergo only a small change in the presence of neighbouring domains and conclude that, provided that care is taken in the choice of domain boundaries, the properties of the titin I-band are essentially "the sum of its parts". From this and other work we propose that variation in kinetic properties between adjacent domains may be a general property of the I-band thereby preventing misfolding events on muscle relaxation.

Mechanical unfolding of a titin Ig domain: structure of transition state revealed by combining atomic force microscopy, protein engineering and molecular dynamics simulations.

Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering Phi-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations in the core do not affect the force required to unfold the protein. As has been shown before, mechanical strength lies in the region of the A' and G-strands but, contrary to previous suggestions, the results indicate clearly that side-chain interactions play a significant role in maintaining mechanical stability. Since Phi-values calculated from molecular dynamics simulations are the same as those determined experimentally, we can, with confidence, use the molecular dynamics simulations to analyse the structure of the transition state in detail, and are able to show loss of interactions between the A' and G-strands with associated A-B and E-F loops in the transition state. The key event is not a simple case of loss of hydrogen bonding interactions between the A' and G-strands alone. Comparison with Phi-values from traditional folding studies shows differences between the force and "no-force" transition states but, nevertheless, the region important for kinetic stability is the same in both cases. This explains the correspondence between hierarchy of kinetic stability (measured in stopped-flow denaturant studies) and mechanical strength in these titin domains.

Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering.

The mechanical unfolding of an immunoglobulin domain from the human muscle protein titin (TI I27) has been shown to proceed via a metastable intermediate in which the A-strand is detached. The structure and properties of this intermediate are characterised in this study. A conservative destabilising mutation in the A-strand has no effect on the unfolding force, nor the dependence of the unfolding force on the pulling speed, indicating that the unfolding forces measured in an AFM experiment are those required for the unfolding of the intermediate and not the native state. A mutant of TI I27 with the A-strand deleted (TI I27-A) is studied by NMR and standard biophysical techniques, combined with protein engineering. Molecular dynamics simulations show TI I27-A to be a good model for the intermediate. It has a structure very similar to the native state, and is surprisingly stable. Comparison with a Phi-value analysis of the unfolding pathway clearly shows that the protein unfolds by a different pathway under an applied force than on addition of denaturant.

Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy.

This manuscript introduces a versatile system for construction of multimeric proteins to be used as substrates for atomic force microscopy. The construction makes use of a cassette system that allows modules to be cut and ligated in any combination in eight different positions. The modules can be sequenced in situ after construction. A three-module fragment can be produced that is of a size amenable to structural and biophysical analysis to check the effect of placing a protein into a multimeric construct. We show that if the parent titin modules are retained in a construct, they can act both as linkers and as an internal standard for the force measurements. Proteins that cannot be expressed solubly in an eight-module homopolymer have been expressed and subject to force measurements using this system.

Evolution of oligomeric state through allosteric pathways that mimic ligand binding.

Evolution and design of protein complexes are almost always viewed through the lens of amino acid mutations at protein interfaces. We showed previously that residues not involved in the physical interaction between proteins make important contributions to oligomerization by acting indirectly or allosterically. In this work, we sought to investigate the mechanism by which allosteric mutations act, using the example of the PyrR family of pyrimidine operon attenuators. In this family, a perfectly sequence-conserved helix that forms a tetrameric interface is exposed as solvent-accessible surface in dimeric orthologs. This means that mutations must be acting from a distance to destabilize the interface. We identified 11 key mutations controlling oligomeric state, all distant from the interfaces and outside ligand-binding pockets. Finally, we show that the key mutations introduce conformational changes equivalent to the conformational shift between the free versus nucleotide-bound conformations of the proteins.