The effect of mutation on an aggregation-prone protein: An in vivo, in vitro, and in silico analysis.

Aggregation of initially stably structured proteins is involved in more than 20 human amyloid diseases. Despite intense research, however, how this class of proteins assembles into amyloid fibrils remains poorly understood, principally because of the complex effects of amino acid substitutions on protein stability, solubility, and aggregation propensity. We address this question using ß2-microglobulin (ß2m) as a model system, focusing on D76N-ß2m that is involved in hereditary amyloidosis. This amino acid substitution causes the aggregation-resilient wild-type protein to become highly aggregation prone in vitro, although the mechanism by which this occurs remained elusive. Here, we identify the residues key to protecting ß2m from aggregation by coupling aggregation with antibiotic resistance in E. coli using a tripartite ß-lactamase assay (TPBLA). By performing saturation mutagenesis at three different sites (D53X-, D76X-, and D98X-ß2m) we show that residue 76 has a unique ability to drive ß2m aggregation in vivo and in vitro. Using a randomly mutated D76N-ß2m variant library, we show that all of the mutations found to improve protein behavior involve residues in a single aggregation-prone region (APR) (residues 60 to 66). Surprisingly, no correlation was found between protein stability and protein aggregation rate or yield, with several mutations in the APR decreasing aggregation without affecting stability. Together, the results demonstrate the power of the TPBLA to develop proteins that are resilient to aggregation and suggest a model for D76N-ß2m aggregation involving the formation of long-range couplings between the APR and Asn76 in a nonnative state.

The role of ß-barrels 1 and 2 in the enzymatic activity of factor XIII A-subunit.

Essentials The roles of ß-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking ß-barrel 2, both ß-barrels, or full length FXIII, were made. Removing ß-barrel 2 caused total loss of activity, removing both ß-barrels returned 30% activity. ß-barrel 2 is necessary for exposure of the active site cysteine during activation. SUMMARY: Background Factor XIII is composed of an activation peptide segment, a ß-sandwich domain, a catalytic core, and, finally, ß-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives To assess the functional roles of ß-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to ß-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to ß-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of ß-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of ß-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the ß-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the ß-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual ß-barrel domains in modulating access to the FXIII active site region.

Advances in ion mobility spectrometry-mass spectrometry reveal key insights into amyloid assembly.

Interfacing ion mobility spectrometry to mass spectrometry (IMS-MS) has enabled mass spectrometric analyses to extend into an extra dimension, providing unrivalled separation and structural characterization of lowly populated species in heterogeneous mixtures. One biological system that has benefitted significantly from such advances is that of amyloid formation. Using IMS-MS, progress has been made into identifying transiently populated monomeric and oligomeric species for a number of different amyloid systems and has led to an enhanced understanding of the mechanism by which small molecules modulate amyloid formation. This review highlights recent advances in this field, which have been accelerated by the commercial availability of IMS-MS instruments. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Specific glycosaminoglycans promote unseeded amyloid formation from beta2-microglobulin under physiological conditions.

Dialysis-related amyloidosis (DRA) is a complication of hemodialysis where beta2-microglobulin (beta2m) forms plaques mainly in cartilaginous tissues. The tissue-specific deposition, along with a known intransigence of pure beta2m to form fibrils in vitro at neutral pH in the absence of preformed fibrillar seeds, suggests a role for factors within cartilage in enhancing amyloid formation from this protein. To identify these factors, we determined the ability of a derivative lacking the N-terminal six amino acids found in ex vivo beta2m amyloid deposits to form amyloid fibrils at pH 7.4 in the absence of fibrillar seeds. We show that the addition of the glycosaminoglycans (GAGs) chrondroitin-4 or 6-sulfate to fibril growth assays results in the spontaneous generation of amyloid-like fibrils. By contrast, no fibrils are observed over the same time course in the presence of hyaluronic acid, a nonsulfated GAG that is abundant in cartilaginous joints. Based on the observation that hyaluronic acid has no effect on fibril stability, while chrondroitin-6-sulfate decreases the rate of fibril disassembly, we propose that the latter GAG enhances amyloid formation by stabilizing the rare fibrils that form spontaneously. This leads to the accumulation of beta2m in fibrillar deposits. Our data rationalize the joint-specific deposition of beta2m amyloid in DRA, suggesting mechanisms by which amyloid formation may be promoted.

Progressive glycosylation of albumin and its effect on the binding of homocysteine may be a key step in the pathogenesis of vascular damage in diabetes mellitus.

The majority of diabetes research to date has rightly focussed on the direct effects of hyperglycaemia on tissues and a number of theories relating to the pathogenesis of vascular disease have been proposed. This research is important as until methods are found to achieve glycaemic control in all diabetic patients, prophylactic interventions to prevent vasculopathy will be required. One of the major blood proteins, human albumin is known to be covalently modified by extended incubation with glucose, leading to an impairment of ligand binding. One of the important ligands bound by albumin is homocysteine. There is increasing and compelling clinical, experimental and epidemiological evidence that homocysteine, and in particular the free unbound fraction, is vasculotoxic. If homocysteine binding to albumin is impaired by increasing glycosylation of albumin then either drugs which reduce homocysteine levels (pyridoxine, folic acid and cobalamin) or inhibit glycosylation (aminoguanidines) may be of benefit in the prevention of vascular damage in diabetic patients.

Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro.

Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils ex vivo are longer and straighter than those previously produced ab initio in vitro. Here we explore the effect of growth conditions on morphology of beta(2)M fibrils formed ab initio in vitro from the wild-type protein, as well as a variant form of beta(2)M in which Asn17 is deamidated to Asp (N17D). We show that deamidation results in significant destabilisation of beta(2)M at neutral pH. Despite this, acidification is still necessary to form amyloid from the mutant protein in vitro. Interestingly, at low pH and low ionic strength long, straight fibrils of recombinant beta(2)M are formed in vitro. The fibrils comprise three distinct morphological types when examined using electron microscopy (EM) and atomic force microscopy (AFM) that vary in periodicity and the number of constituent protofibrils. Using kinetic experiments we suggest that the immature fibrils observed previously do not represent intermediates in the assembly of fully mature amyloid, at least under the conditions studied here.

Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9.

The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins.

Role of the single disulphide bond of beta(2)-microglobulin in amyloidosis in vitro.

The aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils occurs in the condition known as dialysis-related amyloidosis (DRA). The protein has a beta-sandwich fold typical of the immunoglobulin family, which is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized beta(2)m forms amyloid fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single disulphide bond of beta(2)m in amyloidosis in vitro. We show that reduction of the disulphide bond destabilizes the native protein such that non-native molecules are populated at neutral pH. These species are prone to oligomerization but do not form amyloid fibrils when incubated for up to 8 mo at pH 7.0 in 0.4 M NaCl. Over the pH range 4.0-1.5 in the presence of 0.4 M NaCl, however, amyloid fibrils of reduced beta(2)m are formed. These fibrils are approximately 10 nm wide, but are shorter and assemble more rapidly than those produced from the oxidized protein. These data show that population of non-native conformers of beta(2)m at neutral pH by reduction of its single disulphide bond is not sufficient for amyloid formation. Instead, association of one or more specific partially unfolded molecules formed at acid pH are necessary for the formation of beta(2)m amyloid in vitro. Further experiments will now be needed to determine the role of different oligomeric species of beta(2)m in the toxicity of the protein in vivo.

A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding.

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.

Using chimeric immunity proteins to explore the energy landscape for alpha-helical protein folding.

To address the role of sequence in the folding of homologous proteins, the folding and unfolding kinetics of the all-helical bacterial immunity proteins Im2 and Im9 were characterised, together with six chimeric derivatives of these proteins. We show that both Im2 and Im9 fold rapidly (k(UN)(H(2)O)) approximately 2000 s(-1) at pH 7.0, 25 degrees C) in apparent two-state transitions, through rate-limiting transition states that are highly compact (beta(TS)0.93 and 0.96, respectively). Whilst the folding and unfolding properties of three of the chimeras (Im2 (1-44)(Im9), Im2 (1-64)(Im9 )and Im2 (25-44)(Im9)) are similar to their parental counterparts, in other chimeric proteins the introduced sequence variation results in altered kinetic behaviour. At low urea concentrations, Im2 (1-29)(Im9) and Im2 (56-64)(Im9) fold in two-state transitions via transition states that are significantly less compact (beta(TS) approximately 0.7) than those characterised for the other immunity proteins presented here. At higher urea concentrations, however, the rate-limiting transition state for these two chimeras switches or moves to a more compact species (beta(TS) approximately 0.9). Surprisingly, Im2 (30-64)(Im9) populates a highly collapsed species (beta(I)=0.87) in the dead-time (2.5 ms) of stopped flow measurements. These data indicate that whilst topology may place significant constraints on the folding process, specific inter-residue interactions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape.

Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate.

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of approximately 150 micros, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.

Protein folding: progress made and promises ahead.

Over the past 25 years, enormous breakthroughs have been made in understanding protein folding mechanisms. We have now reached an exciting stage, with consensuses beginning to emerge that combine both theoretical and experimental approaches. In addition, new fields have emerged and burgeoned, including in vivo folding and the study of protein misfolding diseases. In today's post-genomic world, understanding protein folding has never been more important and the topic has wide-ranging impact in fields from structural biology to materials science.

Protein folding: pulling back the frontiers.

In the past few years, it has become possible to measure the forces required to mechanically unfold single protein molecules. Recently, the mechanical properties of heteropolyproteins have been studied, shedding new light on the mechanical design of modular proteins such as titin.

Partially unfolded states of beta(2)-microglobulin and amyloid formation in vitro.

Dialysis-related amyloidosis (DRA) involves the aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric beta(2)m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are approximately 10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric beta(2)m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK(a) of 4.7 destabilizes native beta(2)m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional (1)H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for beta(2)m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.

The structure and dynamics in solution of Cu(I) pseudoazurin from Paracoccus pantotrophus.

The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods. The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35+/-0.06 A, using 1,498 distance and 55 torsion angle constraints. The protein has a double-wound Greek-key fold with two alpha-helices toward its C-terminus, similar to that of its oxidized counterpart determined by X-ray crystallography. Comparison of the Cu(I) solution structure with the X-ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements. Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale.

Partially unfolded species populated during equilibrium denaturation of the beta-sheet protein Y74W apo-pseudoazurin.

Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which a unique tryptophan replaces the tyrosine residue found in the tyrosine corner of this Greek key protein, a region that has been proposed to have an important role in folding. Equilibrium denaturation of Y74W apo-pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na(2)SO(4) at 15 degrees C), in which one or more partially folded species are populated in 4. 3 M urea. Using a variety of biophysical techniques, we show that these species, on average, have lost a substantial portion of the native secondary structure, lack fixed tertiary packing involving tryptophan and tyrosine residues, are less compact than the native state as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residual structure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassing this region, however, did not contain detectable nonrandom structure, suggesting that long-range interactions are important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basis of these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates one or more partially unfolded species that are globally collapsed and retain elements of the native structure involving the newly introduced tryptophan residue. We speculate on the role of such intermediates in the generation of the complex Greek key fold.

Ribosome-mediated folding of partially unfolded ricin A-chain.

After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.

Thermal unfolding of an intermediate is associated with non-Arrhenius kinetics in the folding of hen lysozyme.

A variety of techniques, including quenched-flow hydrogen exchange labelling monitored by electrospray ionization mass spectrometry, and stopped-flow absorbance, fluorescence and circular dichroism spectroscopy, has been used to investigate the refolding kinetics of hen lysozyme over a temperature range from 2 degrees C to 50 degrees C. Simple Arrhenius behaviour is not observed, and although the overall rate of folding increases from 2 to 40 degrees C, it decreases above 40 degrees C. In addition, the transient intermediate on the major folding pathway at 20 degrees C, in which the alpha-domain is persistently structured in the absence of a stable beta-domain, is thermally unfolded in a sigmoidal transition (T(m) approximately 40 degrees C) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast ( approximately 25 %) and slow ( approximately 75 %) populations of refolding molecules. By using transition state theory, the kinetic data from various experiments were jointly fitted to a sequential three-state model for the slow folding pathway. Together with previous findings, these results indicate that the alpha-domain intermediate is a productive species on the folding route between the denatured and native states, and which accumulates as a consequence of its intrinsic stability. Our analysis suggests that the temperature dependence of the rate constant for lysozyme folding depends on both the total change in the heat capacity between the ground and transition states (the dominant factor at low temperatures) and the heat-induced destabilization of the alpha-domain intermediate (the dominant factor at high temperatures). Destabilization of such kinetically competent intermediate species is likely to be a determining factor in the non-Arrhenius temperature dependence of the folding rate of those proteins for which one or more intermediates are populated.

Protein folding mechanisms: new methods and emerging ideas.

During the past year, advances in our understanding of folding mechanisms have been made through detailed experimental and theoretical studies of a number of proteins. The development of new methods has allowed the earliest events in folding to be probed and the measurement of folding at the level of individual molecules is now possible, opening the door to exciting new experiments.

GroEL accelerates the refolding of hen lysozyme without changing its folding mechanism.

The chaperonin GroEL binds folding intermediates of four-disulfidehen lysozyme transiently within its central cavity. Using stopped flow fluorescence we show that GroEL binds early intermediates in folding and accelerates the slow kinetic phase that reflects the reversal of non-native interactions involving tryptophan residues and the formation of the native state. Pulsed hydrogen exchange monitored by electrospray ionization mass spectrometry demonstrates that GroEL does not alter the folding mechanism, nor are protected species unfolded by the chaperonin. The data suggest a mechanism for GroEL-assisted folding in which the reorganization of non-native tertiary interactions is facilitated but domain folding is unperturbed.