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

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

A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding.

Although detailed pictures of ribosome structures are emerging, little is known about the structural and cotranslational folding properties of nascent polypeptide chains at the atomic level. Here we used solution-state NMR spectroscopy to define a structural ensemble of a ribosome-nascent chain complex (RNC) formed during protein biosynthesis in Escherichia coli, in which a pair of immunoglobulin-like domains adopts a folded N-terminal domain (FLN5) and a disordered but compact C-terminal domain (FLN6). To study how FLN5 acquires its native structure cotranslationally, we progressively shortened the RNC constructs. We found that the ribosome modulates the folding process, because the complete sequence of FLN5 emerged well beyond the tunnel before acquiring native structure, whereas FLN5 in isolation folded spontaneously, even when truncated. This finding suggests that regulating structure acquisition during biosynthesis can reduce the probability of misfolding, particularly of homologous domains.

Protein unfolding is essential for cleavage within the α-helix of a model protein substrate by the serine protease, thrombin.

Proteolysis has a critical role in transmitting information within a biological system and therefore an important element of biology is to determine the subset of proteins amenable to proteolysis. Until recently, it has been thought that proteases cleave native protein substrates only within solvent exposed loops, but recent evidence indicates that cleavage sites located within α-helices can also be cleaved by proteases, despite the conformation of this secondary structure being generally incompatible with binding into an active site of a protease. In this study, we address the mechanism by which a serine endopeptidase, thrombin, recognizes and cleaves a target sequence located within an α-helix. Thrombin was able to cleave a model substrate, protein G, within its α-helix when a suitable cleavage sequence for the enzyme was introduced into this region. However, structural data for the complex revealed that thrombin was not perturbing the structure of the α-helix, thus it was not destabilizing the helix in order to allow it to fit within its active site. This indicated that thrombin was only cleaving within the α-helix when it was in an unfolded state. In support of this, the introduction of destabilizing mutations within the protein increased the efficiency of cleavage by the enzyme. Our data suggest that a folded α-helix cannot be proteolytically cleaved by thrombin, but the species targeted are the unfolded conformations of the native state ensemble.

A method for the incremental expansion of polyglutamine repeats in recombinant proteins.

The polyglutamine diseases are caused by the expansion of CAG repeats. A key step in understanding the disease mechanisms, at the DNA and protein level, is the ability to produce recombinant proteins with specific length glutamine tracts which is a time-consuming first step in setting up in vitro systems to study the effects of polyglutamine expansion. Described here is a PCR-based method for the amplification of CAG repeats, which we used to incrementally extend CAG length by 3-5 repeats per cycle. This method could be translated into various contexts where amplification of repeating elements is necessary.

Molecular pathways to polyglutamine aggregation.

Over 100 human cellular proteins contain a repetitive polyglutamine tract, however, only nine ofthese proteins are associated with disease. In these proteins, the expanded polyQ tract perturbs the native conformation, resulting in an ordered aggregation process that leads to the formation of amyloid-like fibrils. The misfolding pathway involves the formation of prefibrillar oligomeric structures, which are proposed to be involved in cellular toxicity. Non-polyQ host protein regions modulate the misfolding pathway, suggesting an importance ofprotein context in aggregation. This chapter describes the current research regarding polyQ misfolding, with emphasis on the species populated during aggregation, suggesting an important role of protein context in modulating the aggregation pathway.

Discovery of amino acid motifs for thrombin cleavage and validation using a model substrate.

Understanding the active site preferences of an enzyme is critical to the design of effective inhibitors and to gaining insights into its mechanisms of action on substrates. While the subsite specificity of thrombin is understood, it is not clear whether the enzyme prefers individual amino acids at each subsite in isolation or prefers to cleave combinations of amino acids as a motif. To investigate whether preferred peptide motifs for cleavage could be identified for thrombin, we exposed a phage-displayed peptide library to thrombin. The resulting preferentially cleaved substrates were analyzed using the technique of association rule discovery. The results revealed that thrombin selected for amino acid motifs in cleavage sites. The contribution of these hypothetical motifs to substrate cleavage efficiency was further investigated using the B1 IgG-binding domain of streptococcal protein G as a model substrate. Introduction of a P(2)-P(1)' LRS thrombin cleavage sequence within a major loop of the protein led to cleavage of the protein by thrombin, with the cleavage efficiency increasing with the length of the loop. Introduction of further P(3)-P(1) and P(1)-P(1)'-P(3)' amino acid motifs into the loop region yielded greater cleavage efficiencies, suggesting that the susceptibility of a protein substrate to cleavage by thrombin is influenced by these motifs, perhaps because of cooperative effects between subsites closest to the scissile peptide bond.

The rate of polyQ-mediated aggregation is dramatically affected by the number and location of surrounding domains.

The nine polyglutamine (polyQ) neurodegenerative diseases are caused in part by a gain-of-function mechanism involving protein misfolding, the deposition of ß-sheet-rich aggregates and neuronal toxicity. While previous experimental evidence suggests that the polyQ-induced misfolding mechanism is context dependent, the properties of the host protein, including the domain architecture and location of the polyQ tract, have not been investigated. Here, we use variants of a model polyQ-containing protein to systematically determine the effect of the location and number of flanking folded domains on polyQ-mediated aggregation. Our data indicate that when a pathological-length polyQ tract is present between two domains, it aggregates more slowly than the same-length tract in a terminal location within the protein. We also demonstrate that increasing the number of flanking domains decreases the polyQ protein's aggregation rate. Our experimental data, together with a bioinformatic analysis of all human proteins possessing polyQ tracts, suggest that repeat location and protein domain architecture affect the disease susceptibility of human polyQ proteins.

Flanking domain stability modulates the aggregation kinetics of a polyglutamine disease protein.

Spinocerebellar Ataxia Type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases that are all characterized by progressive neuronal dysfunction and the presence of neuronal inclusions containing aggregated polyQ protein, suggesting that protein misfolding is a key part of this disease. Ataxin-3, the causative protein of SCA3, contains a globular, structured N-terminal domain (the Josephin domain) and a flexible polyQ-containing C-terminal tail, the repeat-length of which modulates pathogenicity. It has been suggested that the fibrillogenesis pathway of ataxin-3 begins with a non-polyQ-dependent step mediated by Josephin domain interactions, followed by a polyQ-dependent step. To test the involvement of the Josephin domain in ataxin-3 fibrillogenesis, we have created both pathogenic and nonpathogenic length ataxin-3 variants with a stabilized Josephin domain, and have both stabilized and destabilized the isolated Josephin domain. We show that changing the thermodynamic stability of the Josephin domain modulates ataxin-3 fibrillogenesis. These data support the hypothesis that the first stage of ataxin-3 fibrillogenesis is caused by interactions involving the non-polyQ containing Josephin domain and that the thermodynamic stability of this domain is linked to the aggregation propensity of ataxin-3.

PolyQ: a database describing the sequence and domain context of polyglutamine repeats in proteins.

The polyglutamine diseases are caused in part by a gain-of-function mechanism of neuronal toxicity involving protein conformational changes that result in the formation and deposition of ß-sheet rich aggregates. Recent evidence suggests that the misfolding mechanism is context-dependent, and that properties of the host protein, including the domain architecture and location of the repeat tract, can modulate aggregation. In order to allow the bioinformatic investigation of the context of polyglutamines, we have constructed a database, PolyQ (http://pxgrid.med.monash.edu.au/polyq). We have collected the sequences of all human proteins containing runs of seven or more glutamine residues and annotated their sequences with domain information. PolyQ can be interrogated such that the sequence context of polyglutamine repeats in disease and non-disease associated proteins can be investigated.

Small heat-shock proteins interact with a flanking domain to suppress polyglutamine aggregation.

Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal cell death in nine diseases, including Huntington disease and the spinocerebellar ataxias (SCAs). There is evidence that sHsps have a role in suppression of polyQ-induced neurodegeneration; for example, the sHsp alphaB-crystallin (alphaB-c) has been identified as a suppressor of SCA3 toxicity in a Drosophila model. However, the molecular mechanism for this suppression is unknown. In this study we tested the ability of alphaB-c to suppress the aggregation of a polyQ protein. We found that alphaB-c does not inhibit the formation of SDS-insoluble polyQ fibrils. We further tested the effect of alphaB-c on the aggregation of ataxin-3, a polyQ protein that aggregates via a two-stage aggregation mechanism. The first stage involves association of the N-terminal Josephin domain followed by polyQ-mediated interactions and the formation of SDS-resistant mature fibrils. Our data show that alphaB-c potently inhibits the first stage of ataxin-3 aggregation; however, the second polyQ-dependent stage can still proceed. By using NMR spectroscopy, we have determined that alphaB-c interacts with an extensive region on the surface of the Josephin domain. These data provide an example of a domain/region flanking an amyloidogenic sequence that has a critical role in modulating aggregation of a polypeptide and plays a role in the interaction with molecular chaperones to prevent this aggregation.

Kinetic instability of the serpin Z alpha1-antitrypsin promotes aggregation.

The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is alpha(1)-antitrypsin (alpha(1)AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of alpha(1)AT, which accounts for 95% of all cases of alpha(1)AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z alpha(1)AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type alpha(1)AT and Z alpha(1)AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type alpha(1)AT and Z alpha(1)AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z alpha(1)AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed.

The structural impact of a polyglutamine tract is location-dependent.

Polyglutamine (polyQ) expansion leads to protein aggregation and neurodegeneration in Huntington's disease and eight other inherited neurological conditions. Expansion of the polyQ tract beyond a threshold of 37 glutamines leads to the formation of toxic nuclear aggregates. This suggests that polyQ expansion causes a conformational change within the protein, the nature of which is unclear. There is a trend in the disease proteins that the polyQ tract is located external to but not within a structured domain. We have created a model polyQ protein in which the repeat location mimics the flexible environment of the polyQ tract in the disease proteins. Our model protein recapitulates the aggregation features observed with the clinical proteins and allows structural characterization. With the use of NMR spectroscopy and a range of biophysical techniques, we demonstrate that polyQ expansion into the pathological range has no effect on the structure, dynamics, and stability of a domain adjacent to the polyQ tract. To explore the clinical significance of repeat location, we engineered a variant of the model protein with a polyQ tract within the domain, a location that does not mimic physiological context, demonstrating significant destabilization and structural perturbation. These different effects highlight the importance of repeat location. We conclude that protein misfolding within the polyQ tract itself is the driving force behind the key characteristics of polyQ disease, and that structural perturbation of flanking domains is not required.

Structural and biochemical characterization of the oxidoreductase NmDsbA3 from Neisseria meningitidis.

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyzes the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Although most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whereas NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3-A resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidized by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

Enhancing the stability and solubility of TEV protease using in silico design.

The ability to rationally increase the stability and solubility of recombinant proteins has long been a goal of biotechnology and has significant implications for biomedical research. Poorly soluble enzymes, for example, result in the need for larger reaction volumes, longer incubation times, and more restricted reaction conditions, all of which increase the cost and have a negative impact on the feasibility of the process. Rational design is achieved here by means of the PoPMuSiC program, which performs in silico predictions of stability changes upon single-site mutations. We have used this program to increase the stability of the tobacco etch virus (TEV) protein. TEV is a 27-kDa nuclear inclusion protease with stringent specificity that is commonly used for the removal of solubility tags during protein purification protocols. However, while recombinant TEV can be produced in large quantities, a limitation is its relatively poor solubility (generally approximately 1 mg/mL), which means that large volumes and often long incubation times are required for efficient cleavage. Following PoPMuSiC analysis of TEV, five variants predicted to be more stable than the wild type were selected for experimental analysis of their stability, solubility, and activity. Of these, two were found to enhance the solubility of TEV without compromising its functional activity. In addition, a fully active double mutant was found to remain soluble at concentrations in excess of 40 mg/mL. This modified TEV appears thus as an interesting candidate to be used in recombinant protein technology.