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

beta-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related beta-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the beta-lactam ring. Class A beta-lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases. The introduction of new beta-lactam compounds, which are meant to be 'beta-lactamase-stable' or beta-lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne beta-lactamase genes or by the acquisition of new genes coding for beta-lactamases with different catalytic properties. On the basis of the X-ray crystallography structures of several class A beta-lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties. However, despite the many available kinetic, structural and mutagenesis data, the factors explaining the diversity of the specificity profiles of class A beta-lactamases and their amazing catalytic efficiency have not been thoroughly elucidated. The detailed understanding of these phenomena constitutes the cornerstone for the design of future generations of antibiotics.

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII ␤-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those ␤-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 ␤-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of ␤-lactamases.

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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