A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, ␤-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N U), where only the native and the denatured states are significantly populated. Thermally-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent T m ‫ס‬ 60-80°C), and display high conformational stabilities (⌬G(H 2 O) ‫ס‬ 30-60 kJ mole −1 ). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications.Keywords: Camel heavy-chain antibodies; protein stability; protein folding; circular dichroism; fluorescence; Fourier transform infrared spectroscopy; surface plasmon resonance; high pressure Article and publication are at

experiments in Fig. 5, the cultured microglia (see Supplementary Figure) that had been preincubated with or without ATP (50 mM) were injected intrathecally in normal rats (see Supplementary Methods for full details). ImmunohistochemistryTransverse L5 spinal cord sections (30 mm) were cut and processed for immunohistochemistry with anti-P2X4R antibody (Alomone). Identification of the type of P2X 4 R-positive cells was performed with the following markers: for microglia, OX42 (Chemicon) and iba1 (a gift from S. Kohsaka); for astrocytes, GFAP (Boehringer Mannheim); for spinal cord neurons, NeuN (Chemicon) and MAP2 (Chemicon). To assess immunofluorescence staining of cells quantitatively, we measured the immunofluorescence intensity of the P2X 4 R or OX42 as the average pixel intensity within each cell (see also Supplementary Methods). Western blottingWestern blot analysis of P2X 4 R expression in the membrane fraction from L4-L6 spinal cord was performed with anti-P2X4R polyclonal antibody (Oncogene) as described in detail in the Supplementary Methods. Microglial cultureRat primary cultured microglia were prepared in accordance with the method described previously 28 . In brief, mixed glial culture was prepared from neonatal Wistar rats and maintained for 10-16 days in DMEM medium with 10% fetal bovine serum. Immediately before experiments, microglia were collected by a gentle shake as the floating cells over the mixed glial culture. The microglia were transferred to coverslips or to Eppendorf tubes for subsequent intrathecal administration. StatisticsStatistical analyses of the results were made with Student's t-test, Student's paired t-test or the Mann-Whitney U-test. Lancet 353, 1959Lancet 353, -1964Lancet 353, (1999. 2. Woolf, C. J. & Salter, M. W. Neuronal plasticity: Increasing the gain in pain. Science 288, 1765Science 288, -1769Science 288, (2000. 3. Bo, X., Zhang, Y., Nassar, M., Burnstock, G. & Schoepfer, R. A P2X purinoceptor cDNA conferring a novel pharmacological profile. FEBS Lett. 375, 129-133 (1995). 4. Buell, G., Lewis, C., Collo, G., North, R. A. & Surprenant, A. An antagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J. 15, 55-62 (1996). 5. Seguela, P., Haghighi, A., Soghomonian, J. J. & Cooper, E. A novel neuronal P2X ATP receptor ion channel with widespread distribution in the brain. J. Neurosci. 16, 448-455 (1996). 6. Soto, F. et al. P2X4: an ATP-activated ionotropic receptor cloned from rat brain. Proc. Natl Acad. Sci. USA 93, 3684-3688 (1996). 7. Wang, C. Z., Namba, N., Gonoi, T., Inagaki, N. & Seino, S. Cloning and pharmacological characterization of a fourth P2X receptor subtype widely expressed in brain and peripheral tissues including various endocrine tissues. Biochem. Biophys. Res. Commun. 220, 196-202 (1996 Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues 1-3 , often with serious pathological consequences. Non-neuropathic systemic...

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The aggregation of the intrinsically disordered protein α-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of α-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to α-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric α-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of α-synuclein. The NbSyn2:α-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of α-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of α-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of α-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo.

Alpha-synuclein (a-Syn) is a key protein involved in Parkinson's disease (PD) pathology. PD is characterized by the loss of dopaminergic neuronal cells in the substantia nigra pars compacta and the abnormal accumulation and aggregation of a-Syn in the form of Lewy bodies and Lewy neurites. More precisely, the aggregation of a-Syn is associated with the dysfunctionality and degeneration of neurons in PD. Moreover, mutations in the SNCA gene, which encodes a-Syn, cause familial forms of PD and are the basis of sporadic PD risk. Given the role of the a-Syn protein in the pathology of PD, animal models that reflect the dopaminergic neuronal loss and the widespread and progressive formation of a-Syn aggregates in different areas of the brain constitute a valuable tool. Indeed, animal models of PD are important for understanding the molecular mechanisms of the disease and might contribute to the development and validation of new therapies. In the absence of animal models that faithfully reproduce human PD, in recent years, numerous animal models of PD based on a-Syn have been generated. In this review, we summarize the main features of the a-Syn pre-formed fibrils (PFFs) model and recombinant adenoassociated virus vector (rAAV) mediated a-Syn overexpression models, providing a detailed comparative analysis of both models. Here, we discuss how each model has contributed to our understanding of PD pathology and the advantages and weakness of each of them. Significance: Here, we show that injection of a-Syn PFFs and overexpression of a-Syn mediated by rAAV lead to a different pattern of PD pathology in rodents. First, a-Syn PFFs models trigger the Lewy body-like inclusions formation in brain regions directly interconnected with the injection site, suggesting that there is an inter-neuronal transmission of the a-Syn pathology. In contrast, rAAV-mediated a-Syn overexpression in the brain limits the a-Syn aggregates within the transduced neurons. Second, phosphorylated a-Syn inclusions obtained with rAAV are predominantly nuclear with a punctate appearance that becomes diffuse along the neuronal fibers, whereas a-Syn PFFs models lead to the formation of cytoplasmic aggregates of phosphorylated a-Syn reminiscent of Lewy bodies and Lewy neurites.

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fouriertransform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive β-sheet structure and a substantial element of non β-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the β-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.