Evidence for synergistic action of transthyretin and IGF-I over the IGF-I receptor.

Transthyretin (TTR) has a neuroprotective role in the central nervous system (CNS) in Alzheimer's disease (AD) and cerebral ischemia. Increased levels of TTR and activated insulin-like growth factor I receptor (IGF-IR) are associated with reduced neurodegeneration in an AD mouse model. In the present study, we found that TTR and IGF-I have a synergistic effect on activation of one of the IGF-IR signaling pathways. Hippocampus of TTR null mice present decreased levels of phosphorylated IGF-IR and Akt when compared with TTR wild type littermate animals. Cell studies reveal the synergistic effect of TTR and IGF-I in promoting IGF-IR signaling even under glutamate induced toxicity. TTR:IGF-IR complexes are identified and a bio-layer interferometry assay demonstrated an interaction between TTR and IGF-IR with a KD ranging from 99 to 744nM. In summary, our results point to a new TTR role through the IGF-I axis, mediated through TTR-IGF-IR interactions.

Calcium binding to gatekeeper residues flanking aggregation-prone segments underlies non-fibrillar amyloid traits in superoxide dismutase 1 (SOD1).

Calcium deregulation is a central feature among neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Calcium accumulates in the spinal and brain stem motor neurons of ALS patients triggering multiple pathophysiological processes which have been recently shown to include direct effects on the aggregation cascade of superoxide dismutase 1 (SOD1). SOD1 is a Cu/Zn enzyme whose demetallated form is implicated in ALS protein deposits, contributing to toxic gain of function phenotypes. Here we undertake a combined experimental and computational study aimed at establishing the molecular details underlying the regulatory effects of Ca(2+) over SOD1 aggregation potential. Isothermal titration calorimetry indicates entropy driven low affinity association of Ca(2+) ions to apo SOD1, at pH7.5 and 37°C. Molecular dynamics simulations denote a noticeable loss of native structure upon Ca(2+) association that is especially prominent at the zinc-binding and electrostatic loops, whose decoupling is known to expose the central SOD1 ß-barrel triggering aggregation. Structural mapping of the preferential apo SOD1 Ca(2+) binding locations reveals that among the most frequent ligands for Ca(2+) are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. Calcium interactions thus diminish gatekeeping roles of these residues, by shielding repulsive interactions via stacking between aggregating ß-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in ALS inclusions. Interestingly, many fALS mutations occur at these positions, disclosing how Ca(2+) interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic ALS pathomechanisms.

Calcium ions promote superoxide dismutase 1 (SOD1) aggregation into non-fibrillar amyloid: a link to toxic effects of calcium overload in amyotrophic lateral sclerosis (ALS)?

Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca(2+) dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca(2+). We show that at physiological pH, Ca(2+) induces conformational changes that increase SOD1 ß-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca(2+) boosts the onset of SOD1 aggregation. In agreement, Ca(2+) decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca(2+) induced aggregates consisting preferentially of antiparallel ß-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca(2+)-induced aggregates, thus indicating that Ca(2+) diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca(2+) levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca(2+) may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.

S100A6 amyloid fibril formation is calcium-modulated and enhances superoxide dismutase-1 (SOD1) aggregation.

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H(I) and H(IV). Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca(2+) exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca(2+) promotes anti-parallel ß-sheet conformations that repress fibrillation. At pH 7, Ca(2+) rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca(2+). Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

Small molecules present in the cerebrospinal fluid metabolome influence superoxide dismutase 1 aggregation.

Superoxide dismutase 1 (SOD1) aggregation is one of the pathological markers of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. The underlying molecular grounds of SOD1 pathologic aggregation remains obscure as mutations alone are not exclusively the cause for the formation of protein inclusions. Thus, other components in the cell environment likely play a key role in triggering SOD1 toxic aggregation in ALS. Recently, it was found that ALS patients present a specific altered metabolomic profile in the cerebrospinal fluid (CSF) where SOD1 is also present and potentially interacts with metabolites. Here we have investigated how some of these small molecules affect apoSOD1 structure and aggregation propensity. Our results show that as co-solvents, the tested small molecules do not affect apoSOD1 thermal stability but do influence its tertiary interactions and dynamics, as evidenced by combined biophysical analysis and proteolytic susceptibility. Moreover, these compounds influence apoSOD1 aggregation, decreasing nucleation time and promoting the formation of larger and less soluble aggregates, and in some cases polymeric assemblies apparently composed by spherical species resembling the soluble native protein. We conclude that some components of the ALS metabolome that shape the chemical environment in the CSF may influence apoSOD1 conformers and aggregation.

Structural requirements for VAP-B oligomerization and their implication in amyotrophic lateral sclerosis-associated VAP-B(P56S) neurotoxicity.

The integral endoplasmic reticulum (ER)-membrane protein VAP-B interacts with various lipid-transfer/binding proteins containing an FFAT motif through its N-terminal MSP domain. A genetic mutation within its MSP domain, P56S, was identified in familial forms of motor neuron diseases. This mutation induces the formation of insoluble VAP-B(P56S) protein aggregates by an unknown mechanism. In this study, we defined the structural requirements for VAP-B oligomerization and demonstrated their contribution for VAP-B(P56S) aggregation and neurotoxicity. We show that the oligomerization of VAP-B is mainly mediated by its coiled-coil domain and that the GXXXG dimerization motif within the transmembrane domain mediates transmembrane domains self-association but is insufficient to drive VAP-B oligomerization. We further show that the oligomerization of the wild-type VAP-B is independent of its MSP domain. However, we found that the P56S mutation induces conformational changes within the MSP domain and facilitates its propensity to aggregate by exposing hydrophobic patches to the solvent. These conformational changes have no direct effect on FFAT binding. Rather, they enhance VAP-B(P56S) oligomerization driven by the combined contributions of the coiled-coil and the transmembrane domains, thereby preventing accessibility to FFAT-binding site, facilitating the production of VAP-B(P56S)-insoluble aggregates and consequently its neurotoxicity. These results shed light on the mechanism by which VAP-B(P56S) aggregates are formed and induce familial motor neuron diseases.

The prominent conformational plasticity of lactoperoxidase: a chemical and pH stability analysis.

Lactoperoxidase (LPO) is a structurally complex and stable mammalian redox enzyme. Here we aim at evaluating the influence of ionic interactions and how these intertwine with the structural dynamics, stability and activity of LPO. In this respect, we have compared LPO guanidinium hydrochloride (GdmCl) and urea denaturation pathways and performed a detailed investigation on the effects of pH on the LPO conformational dynamics and stability. Our experimental findings using far-UV CD, Trp fluorescence emission and ESR spectroscopies clearly indicate that LPO charged-denaturation with GdmCl induced a sharp two-step process versus a three-step unfolding mechanism induced by urea. This differential effect between GdmCl and urea suggests that ionic interactions must play a rather prominent role in the stabilization of LPO. With both denaturants, the protein core was shown to retain activity up to near the respective C(m) values. Moreover, a pH titration of LPO evidenced no significant conformational alterations or perturbation of heme activity within the 4 to 11 pH interval. In contrast, alterations of ionic interactions by poising LPO at pH 3, 2 and 12 resulted in a loss of secondary structure, loosening of tertiary contacts and loss of activity, which appear to be associated with the perturbation of the hydrophobic core, as evidenced by ANS binding, as well as disruption of the heme pocket demonstrated by optical and EPR spectroscopies. Overall, LPO is characterised by a high degree of peripheral structural plasticity without perturbation of the core heme moiety. The possible physiological meaning of such features is discussed.

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins.

Rieske proteins and Rieske ferredoxins are present in the three domains of life and are involved in a variety of cellular processes. Despite their functional diversity, these small Fe-S proteins contain a highly conserved all-beta fold, which harbors a [2Fe-2S] Rieske center. We have identified a novel subtype of Rieske ferredoxins present in hyperthermophilic archaea, in which a two-cysteine conserved SKTPCX((2-3))C motif is found at the C-terminus. We establish that in the Acidianus ambivalens representative, Rieske ferredoxin 2 (RFd2), these cysteines form a novel disulfide bond within the Rieske fold, which can be selectively broken under mild reducing conditions insufficient to reduce the [2Fe-2S] cluster or affect the secondary structure of the protein, as shown by visible circular dichroism, absorption, and attenuated total reflection Fourier transform IR spectroscopies. RFd2 presents all the EPR, visible absorption, and visible circular dichroism spectroscopic features of the [2Fe-2S] Rieske center. The cluster has a redox potential of +48 mV (25 degrees C and pH 7) and a pK (a) of 10.1 +/- 0.2. These shift to +77 mV and 8.9 +/- 0.3, respectively, upon reduction of the disulfide. RFd2 has a melting temperature near the boiling point of water (T(m) = 99 degrees C, pH 7.0), but it becomes destabilized upon disulfide reduction (DeltaT(m) = -9 degrees C, DeltaC(m) = -0.7 M guanidinium hydrochloride). This example illustrates how the incorporation of an additional structural element such as a disulfide bond in a highly conserved fold such as that of the Rieske domain may fine-tune the protein for a particular function or for increased stability.

On the relative contribution of ionic interactions over iron-sulfur clusters to ferredoxin stability.

Metal centres play an important structural role in maintaining the native conformation of a protein. Here we use biophysical methods to investigate what is the relative contribution of iron-sulfur clusters in respect to ionic interactions in a thermophilic di-cluster ferredoxin model. Changes in protonation affect both the stability and the conformational dynamics of the protein fold. In the pH 5.5-8 interval, the protein has a high melting temperature (T(m) approximately 120 degrees C), which decreases towards pH extremes. Acidification triggers events in two steps: down to the isoelectric point (pH 3.5) the Fe-S clusters remain unchanged, the secondary structure content increases and the single Trp becomes more solvent shielded, denoting a more compact fold. Further acidification down to pH 2 sets off exposure of the hydrophobic core and Fe-S cluster disintegration, yielding a molten globule state. The relative stabilising contribution of the clusters becomes evident when stabilising ionic interactions are switched off as a result of poising the protein at pH 3.5, at an overall null charge: under these conditions, the Fe-S clusters disassemble at T(m)=72 degrees C, whereas the protein unfolds at T(m)=52 degrees C. Overall, this ferredoxin denotes a considerable structural plasticity around its native conformation, a property which appears to depend more on the integrity of its metal clusters rather than on the status of its stabilising electrostatic interactions. The latter however play a relevant role in determining the protein thermal stability.

Lactoperoxidase folding and catalysis relies on the stabilization of the alpha-helix rich core domain: a thermal unfolding study.

Lactoperoxidase (LPO) belongs to the mammalian peroxidase family and catalyzes the oxidation of halides, pseudo-halides and a number of aromatic substrates at the expense of hydrogen peroxide. Despite the complex physiological role of LPO and its potential involvement in carcinogenic mechanisms, cystic fibrosis and inflammatory processes, little is known on the folding and structural stability of this protein. We have undertaken an investigation of the conformational dynamics and catalytic properties of LPO during thermal unfolding, using complementary biophysical techniques (differential scanning calorimetry, electron spin resonance, optical absorption, fluorescence and circular dichroism spectroscopies) together with biological activity assays. LPO is a particularly stable protein, capable of maintaining catalysis and structural integrity up to a high temperature, undergoing irreversible unfolding at 70 degrees C. We have observed that the first stages of the thermal denaturation involve a minor conformational change occurring at 40 degrees C, possibly at the level of the protein beta-sheets, which nevertheless does not result in an unfolding transition. Only at higher temperature, the protein hydrophobic core, which is rich in alpha-helices, unfolds with concomitant disruption of the catalytic heme pocket and activity loss. Evidences concerning the stabilizing role of the disulfide bridges and the covalently bound heme cofactor are shown and discussed in the context of understanding the structural stability determinants in a relatively large protein.

Crystallographic analysis of the intact metal centres [3Fe-4S](1+/0) and [4Fe-4S](2+/1+) in a Zn(2+) -containing ferredoxin.

Detailed structural models of di-cluster seven-iron ferredoxins constitute a valuable resource for folding and stability studies relating the metal cofactors' role in protein stability. The here reported, hemihedric twinned crystal structure at 2.0 A resolution from Acidianus ambivalens ferredoxin, shows an integral 103 residues, physiologically relevant native form composed by a N-terminal extension comprising a His/Asp Zn(2+) site and the ferredoxin (betaalphabeta)(2) core, which harbours intact clusters I and II, a [3Fe-4S](1+/0) and a [4Fe-4S](2+/1+) centres. This is in contrast with the previously available ferredoxin structure from Sulfolofus tokodai, which was obtained from an artificial oxidative conversion with two [3Fe-4S](1+/0) centres and poor definition around cluster II.

The neuronal S100B protein is a calcium-tuned suppressor of amyloid-ß aggregation.

Amyloid-ß (Aß) aggregation and neuroinflammation are consistent features in Alzheimer's disease (AD) and strong candidates for the initiation of neurodegeneration. S100B is one of the most abundant proinflammatory proteins that is chronically up-regulated in AD and is found associated with senile plaques. This recognized biomarker for brain distress may, thus, play roles in amyloid aggregation which remain to be determined. We report a novel role for the neuronal S100B protein as suppressor of Aß42 aggregation and toxicity. We determined the structural details of the interaction between monomeric Aß42 and S100B, which is favored by calcium binding to S100B, possibly involving conformational switching of disordered Aß42 into an α-helical conformer, which locks aggregation. From nuclear magnetic resonance experiments, we show that this dynamic interaction occurs at a promiscuous peptide-binding region within the interfacial cleft of the S100B homodimer. This physical interaction is coupled to a functional role in the inhibition of Aß42 aggregation and toxicity and is tuned by calcium binding to S100B. S100B delays the onset of Aß42 aggregation by interacting with Aß42 monomers inhibiting primary nucleation, and the calcium-bound state substantially affects secondary nucleation by inhibiting fibril surface-catalyzed reactions through S100B binding to growing Aß42 oligomers and fibrils. S100B protects cells from Aß42-mediated toxicity, rescuing cell viability and decreasing apoptosis induced by Aß42 in cell cultures. Together, our findings suggest that molecular targeting of S100B could be translated into development of novel approaches to ameliorate AD neurodegeneration.

Studies of the molten globule state of ferredoxin: structural characterization and implications on protein folding and iron-sulfur center assembly.

The biological insertion of iron-sulfur clusters (Fe-S) involves the interaction of (metallo) chaperons with a partly folded target polypeptide. In this respect, the study of nonnative protein conformations in iron-sulfur proteins is relevant for the understanding of the folding process and cofactor assembly. We have investigated the formation of a molten globule state in the [3Fe4S][4Fe4S] ferredoxin from the thermophilic archaeon Acidianus ambivalens (AaFd), which also contains a structural zinc site. Biophysical studies have shown that, at acidic pH, AaFd retains structural folding and metal centers. However, upon increasing the temperature, a series of successive modifications occur within the protein structure: Fe-S disassembly, loss of tertiary contacts and dissociation of the Zn(2+) site, which is simultaneous to alterations on the secondary structure. Upon cooling, an apo-ferredoxin state is obtained, with characteristics of a molten globule: compactness identical to the native form; similar secondary structure evidenced by far-UV CD; no near-UV CD detected tertiary contacts; and an exposure of the hydrophobic surface evidenced by 1-anilino naphthalene-8-sulfonic acid (ANS) binding. In contrast to the native form, this apo ferredoxin state undergoes reversible thermal and chemical unfolding. Its conformational stability was investigated by guanidinium chloride denaturation and this state is approximately 1.5 kcal mol(-1) destabilised in respect to the holo ferredoxin. The single tryptophan located nearby the Fe-S pocket probed the conformational dynamics of the molten globule state: fluorescence quenching, red edge emission shift analysis and resonance energy transfer to bound ANS evidenced a restricted mobility and confinement within a hydrophobic environment. The possible physiological relevance of molten globule states in Fe-S proteins and the hypothesis that their structural flexibility may be important to the understanding of metal center insertion are discussed.

Characterization of plasma labile heme in hemolytic conditions.

Extracellular hemoglobin, a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here, we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches, we demonstrate that when generated during hemolytic conditions labile heme is bound to plasma molecules with an affinity higher than 10-7 m and that 2-8% (~ 2-5 µm) of the total amount of heme detected in plasma can be internalized by bystander cells, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme-binding capacity, that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10-7 m. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme.

The sulphur oxygenase reductase from Acidianus ambivalens is a multimeric protein containing a low-potential mononuclear non-haem iron centre.

The SOR (sulphur oxygenase reductase) is the initial enzyme in the sulphur-oxidation pathway of Acidianus ambivalens. Expression of the sor gene in Escherichia coli resulted in active, soluble SOR and in inclusion bodies from which active SOR could be refolded as long as ferric ions were present in the refolding solution. Wild-type, recombinant and refolded SOR possessed indistinguishable properties. Conformational stability studies showed that the apparent unfolding free energy in water is approx. 5 kcal x mol(-1) (1 kcal=4.184 kJ), at pH 7. The analysis of the quaternary structures showed a ball-shaped assembly with a central hollow core probably consisting of 24 subunits in a 432 symmetry. The subunits form homodimers as the building blocks of the holoenzyme. Iron was found in the wild-type enzyme at a stoichiometry of one iron atom/subunit. EPR spectroscopy of the colourless SOR resulted in a single isotropic signal at g=4.3, characteristic of high-spin ferric iron. The signal disappeared upon reduction with dithionite or incubation with sulphur at elevated temperature. Thus both EPR and chemical analysis indicate the presence of a mononuclear iron centre, which has a reduction potential of -268 mV at pH 6.5. Protein database inspection identified four SOR protein homologues, but no other significant similarities. The spectroscopic data and the sequence comparison led to the proposal that the Acidianus ambivalens SOR typifies a new type of non-haem iron enzyme containing a mononuclear iron centre co-ordinated by carboxylate and/or histidine ligands.

Calcium dysregulation links ALS defective proteins and motor neuron selective vulnerability.

More than 20 distinct gene loci have so far been implicated in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by progressive neurodegeneration of motor neurons (MN) and death. Most of this distinct set of ALS-related proteins undergoes toxic deposition specifically in MN for reasons which remain unclear. Here we overview a recent body of evidence indicative that mutations in ALS-related proteins can disrupt fundamental Ca(2+) signalling pathways in MN, and that Ca(2+) itself impacts both directly or indirectly in many ALS critical proteins and cellular processes that result in MN neurodegeneration. We argue that the inherent vulnerability of MN to dysregulation of intracellular Ca(2+) is deeply associated with discriminating pathogenicity and aberrant crosstalk of most of the critical proteins involved in ALS. Overall, Ca(2+) deregulation in MN is at the cornerstone of different ALS processes and is likely one of the factors contributing to the selective susceptibility of these cells to this particular neurodegenerative disease.

Aberrant zinc binding to immature conformers of metal-free copper-zinc superoxide dismutase triggers amorphous aggregation.

Superoxide dismutase 1 (SOD1) is a Cu/Zn metalloenzyme that aggregates in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Correct metal insertion during SOD1 biosynthesis is critical to prevent misfolding; however Zn(2+) can bind to the copper-site leading to an aberrantly metallated protein. These effects of Zn(2+) misligation on SOD1 aggregation remain to be explored, even though Zn(2+) levels are upregulated in ALS motor neurons. Here we use complementary biophysical methods to investigate Zn(2+) binding and its effects on the aggregation of three immature metal-free SOD1 conformers that represent biogenesis intermediates: dimeric, monomeric and reduced monomeric SOD1. Using isothermal titration calorimetry we determined that Zn(2+) binds to all conformers both at the zinc- as well as to the copper-site; however Zn(2+) binding mechanisms to the zinc-site have distinct characteristics across immature conformers. We show that this 'zinc overload' of immature SOD1 promotes intermolecular interactions, as evidenced by dynamic light scattering and ThT fluorescence kinetic studies. Analysis of aged zinc-induced aggregates by energy-dispersive X-ray and electron energy-loss spectroscopy shows that aggregates integrate some Zn(2+). In addition, electron diffraction analysis identifies nano-scaled crystalline materials and amyloid fibril-like reflections. Transmission electron microscopy reveals that Zn(2+) diverts the SOD1 aggregation pathway from fibrils to amorphous aggregate, and electrophoretic analysis evidences an increase in insoluble materials. Overall, we provide evidence that aberrant zinc coordination to immature conformers broadens the population of SOD1 misfolded species at early aggregation stages and provide evidence for a high structural polymorphism and heterogeneity of SOD1 aggregates.

Intrinsically disordered and aggregation prone regions underlie ß-aggregation in S100 proteins.

S100 proteins are small dimeric calcium-binding proteins which control cell cycle, growth and differentiation via interactions with different target proteins. Intrinsic disorder is a hallmark among many signaling proteins and S100 proteins have been proposed to contain disorder-prone regions. Interestingly, some S100 proteins also form amyloids: S100A8/A9 forms fibrils in prostatic inclusions and S100A6 fibrillates in vitro and seeds SOD1 aggregation. Here we report a study designed to investigate whether ß-aggregation is a feature extensive to more members of S100 family. In silico analysis of seven human S100 proteins revealed a direct correlation between aggregation and intrinsic disorder propensity scores, suggesting a relationship between these two independent properties. Averaged position-specific analysis and structural mapping showed that disorder-prone segments are contiguous to aggregation-prone regions and that whereas disorder is prominent on the hinge and target protein-interaction regions, segments with high aggregation propensity are found in ordered regions within the dimer interface. Acidic conditions likely destabilize the seven S100 studied by decreasing the shielding of aggregation-prone regions afforded by the quaternary structure. In agreement with the in silico analysis, hydrophobic moieties become accessible as indicated by strong ANS fluorescence. ATR-FTIR spectra support a structural inter-conversion from α-helices to intermolecular ß-sheets, and prompt ThT-binding takes place with no noticeable lag phase. Dot blot analysis using amyloid conformational antibodies denotes a high diversity of conformers; subsequent analysis by TEM shows fibrils as dominant species. Altogether, our data suggests that ß-aggregation and disorder-propensity are related properties in S100 proteins, and that the onset of aggregation is likely triggered by loss of protective tertiary and quaternary interactions.

A spectroscopic study of the temperature induced modifications on ferredoxin folding and iron-sulfur moieties.

Thermal perturbation of the dicluster ferredoxin from Acidianus ambivalens was investigated employing a toolbox of spectroscopic methods. FTIR and visible CD were used for assessing changes of the secondary structure and coarse alterations of the [3Fe4S] and [4Fe4S] cluster moieties, respectively. Fine details of the disassembly of the metal centers were revealed by paramagnetic NMR and resonance Raman spectroscopy. Overall, thermally induced unfolding of AaFd is initiated with the loss of -helical content at relatively low temperatures (T(app)(m) approximately 44 degrees C), followed by the disruption of both iron-sulfur clusters (T(app)(m) approximately 53-60 degrees C). The degradation of the metal centers triggers major structural changes on the protein matrix, including the loss of tertiary contacts (T(app)(m) approximately 58 degrees C) and a change, rather than a significant net loss, of secondary structure (T(app)(m) approximately 60 degrees C). This latter process triggers a secondary structure reorganization that is consistent with the formation of a molten globule state. The combined spectroscopic approach here reported illustrates how changes in the metalloprotein organization are intertwined with disassembly of the iron-sulfur centers, denoting the conformational interplay of the protein backbone with cofactors.

Natural domain design: enhanced thermal stability of a zinc-lacking ferredoxin isoform shows that a hydrophobic core efficiently replaces the structural metal site.

Zinc centers play a key role as important structure determinants in a variety of proteins including ferredoxins (Fd). Here, we exploit the availability of two highly similar ferredoxin isoforms from the thermophile Sulfolobus metallicus, which differ in the residues involved in coordinating a His/Asp zinc site that ties together the protein core with its N-terminal extension, to investigate the effect of the absence of this site on ferredoxin folding. The conformational properties of the zinc-containing (FdA) and zinc-lacking (FdB) isoforms were investigated using visible absorption and tryptophan fluorescence emission. Fluorescence quenching studies, together with comparative modeling and molecular dynamics simulations, indicate that the FdB N-terminal extension assumes a fold identical to that of the Zn(2+)-containing isoform. The thermal stability of the isoforms was investigated in a broad pH range (2 < pH < 10), and at physiological pH conditions, both proteins unfold above 100 degrees C. Surprisingly, the Zn(2+)-lacking isoform was always found to be more stable than its Zn(2+)-containing counterpart: a DeltaT(m) approximately 9 degrees C is determined at pH 7, a difference that becomes even more significant at extreme pH values, reaching a DeltaT(m) approximately 24 degrees C at pH 2 and 10. The contribution of the Zn(2+) site to ferredoxin stability was further resolved using selective metal chelators. During thermal unfolding, the zinc scavenger TPEN significantly lowers the T(m) in FdA ( approximately 10 degrees C), whereas it has no effect in FdB. This shows that the Zn(2+) site contributes to ferredoxin stability but that FdB has devised a structural strategy that accounts for an enhanced stability without using a metal cross-linker. An analysis of the FdB sequence and structural model leads us to propose that the higher stability of the zinc-containing ferredoxin results from van der Waals contacts formed between the residues that occupy the same spatial region where the zinc ligands are found in FdA. These favor the formation of a novel local stabilizing hydrophobic core and illustrate a strategy of natural fold design.