Probing the folding pathway of a consensus serpin using single tryptophan mutants.

Conserpin is an engineered protein that represents the consensus of a sequence alignment of eukaryotic serpins: protease inhibitors typified by a metastable native state and a structurally well-conserved scaffold. Previously, this protein has been found to adopt a native inhibitory conformation, possess an atypical reversible folding pathway and exhibit pronounced resistance to inactivation. Here we have designed a version of conserpin, cAT, with the inhibitory specificity of α-antitrypsin, and generated single-tryptophan variants to probe its folding pathway in more detail.

Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells.

Amyloidogenic protein aggregation impairs cell function and is a hallmark of many chronic degenerative disorders. Protein aggregation is also a major event during acute injury; however, unlike amyloidogenesis, the process of injury-induced protein aggregation remains largely undefined. To provide this insight, we profiled the insoluble proteome of several cell types after acute injury.

Smoothing a rugged protein folding landscape by sequence-based redesign.

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases.

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.

Enhanced molecular mobility of ordinarily structured regions drives polyglutamine disease.

Polyglutamine expansion is a hallmark of nine neurodegenerative diseases, with protein aggregation intrinsically linked to disease progression. Although polyglutamine expansion accelerates protein aggregation, the misfolding process is frequently instigated by flanking domains. For example, polyglutamine expansion in ataxin-3 allosterically triggers the aggregation of the catalytic Josephin domain.

The Z mutation alters the global structural dynamics of α1-antitrypsin.

α1-Antitrypsin (α1AT) deficiency, the most common serpinopathy, results in both emphysema and liver disease. Over 90% of all clinical cases of α1AT deficiency are caused by the Z variant in which Glu342, located at the top of s5A, is replaced by a Lys which results in polymerization both in vivo and in vitro. The Glu342Lys mutation removes a salt bridge and a hydrogen bond but does not effect the thermodynamic stability of Z α1AT compared to the wild type protein, M α1AT, and so it is unclear why Z α1AT has an increased polymerization propensity.

Oxidation of an exposed methionine instigates the aggregation of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of "nucleocytoplasmic coagulation." Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation.

Conformational behavior and aggregation of ataxin-3 in SDS.

Spinocerebellar ataxia type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases all characterized by the presence of intraneuronal inclusions that contain aggregated protein. Aggregation of ataxin-3, the causative protein of SCA3, has been well characterized in vitro, with both pathogenic and non-pathogenic length ataxin-3 undergoing fibrillogenesis. However, only ataxin-3 containing an expanded polyQ tract leads to SCA3.

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.

The roles of helix I and strand 5A in the folding, function and misfolding of α1-antitrypsin.

α(1)-Antitrypsin, the archetypal member of the serpin superfamily, is a metastable protein prone to polymerization when exposed to stressors such as elevated temperature, low denaturant concentrations or through the presence of deleterious mutations which, in a physiological context, are often associated with disease. Experimental evidence suggests that α(1)-Antitrypsin can polymerize via several alternative mechanisms in vitro. In these polymerization mechanisms different parts of the molecule are proposed to undergo conformational change.

Nucleocytoplasmic coagulation: an injury-induced aggregation event that disulfide crosslinks proteins and facilitates their removal by plasmin.

Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. In vitro and in vivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers.

Conformational properties of the disease-causing Z variant of α1-antitrypsin revealed by theory and experiment.

The human serine protease inhibitor (serpin) α-1 antitrypsin (α1-AT) protects tissues from proteases of inflammatory cells. The most common disease-causing mutation in α1-AT is the Z-mutation (E342K) that results in an increased propensity of α1-AT to polymerize in the ER of hepatocytes, leading to a lack of secretion into the circulation. The structural consequences of this mutation, however, remain elusive.

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.

Methods to measure the kinetics of protease inhibition by serpins.

The serpin molecule has evolved an unusual mechanism of inhibition, involving an exposed reactive center loop (RCL) and conformational change to covalently trap a target protease. Successful inhibition of the protease is dependent on the rate of serpin-protease association and the efficiency with which the RCL inserts into β-sheet A, translocating the covalently bound protease and thereby completing the inhibition process. This chapter describes the kinetic methods used for determining the rate of protease inhibition (k(a)) and the stoichiometry of inhibition.

Intracellular production of recombinant serpins in yeast.

Yeast are a valuable system for recombinant serpin production due to their ability to synthesize large amounts of heterologous gene products as well as their expression of folding chaperones and lack of endogenous serpin genes. In this chapter, we describe a method for intracellular expression of cytoplasmic serpins in the yeast Pichia pastoris. We also give details on how this system can be exploited to produce polymer-forming mutants of secretory serpins.

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 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 β-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.

Structural change in β-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease.

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α(1)-antitrypsin (α(1)AT) deficiency cases. It leads to increased polymerization of the serpin α(1)AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization.

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.

Refolding your protein with a little help from REFOLD.

The expression and harvesting of proteins from insoluble inclusion bodies by solubilization and refolding is a technique commonly used in the production of recombinant proteins. Despite the importance of refolding, publications in the literature are essentially ad hoc reports consisting of a dazzling array of experimental protocols and a diverse collection of buffer cocktails. For the protein scientists, using this information to refold their protein of interest presents enormous challenges.

Amyloid formation from an α-helix peptide bundle is seeded by 3(10)-helix aggregates.

Transformation of proteins and peptides to fibrillar aggregates rich in β sheets underlies many diseases, but mechanistic details of these structural transitions are poorly understood. To simulate aggregation, four equivalents of a water-soluble, α-helical (65 %) amphipathic peptide (AEQLLQEAEQLLQEL) were assembled in parallel on an oxazole-containing macrocyclic scaffold. The resulting 4α-helix bundle is monomeric and even more α helical (85 %), but it is also unstable at pH 4 and undergoes concentration-dependent conversion to β-sheet aggregates and amyloid fibrils.

The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D.

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 β-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.