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

We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C).

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Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins, induces folding, and counteracts the denaturing effects of urea, pressure, and ice. To establish the mechanism behind these effects, isotopic substitution neutron-scattering measurements were performed on aqueous solutions of TMAO and 1:1 TMAO-urea at a solute mole fraction of 0.05. The partial pair distribution functions were extracted using the empirical potential structure refinement method. The results were compared with previous results obtained with isosteric tert-butanol, as well as the available data from spectroscopy and molecular-dynamics simulations. In solution, the oxygen atom of TMAO is strongly hydrogen-bonded to, on average, between two and three water molecules, and the hydrogen-bond network is tighter in water than in pure water. In TMAO-urea solutions, the oxygen atom in TMAO preferentially forms hydrogen bonds with urea. This explains why the counteraction is completed at a 2:1 urea/TMAO concentration ratio, independently of urea concentration. These results strongly support models for the effect of TMAO on the stability of proteins based on a modification of the simultaneous equilibria that control hydrogen bonding between the peptide backbone and water or intramolecular sites, without any need for direct interaction between TMAO and the protein.

Endochondral ossification, an important bone formation process in vertebrates, highly depends on proper functioning of growth plate chondrocytes 1 . Their proliferation determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment 1,2 . In addition, the centre of the expanding growth plate becomes hypoxic and local activation of the hypoxiainducible transcription factor HIF-1α is necessary for chondrocyte survival by still unknown cellintrinsic mechanisms [3][4][5][6] . Whether HIF-1α signalling has to be contained in the other regions of the growth plate and whether chondrocyte metabolism controls cell function remains undefined. We here show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response (UPR) and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate (αKG) levels, which in turn increases collagen proline and lysine hydroxylation. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to proteasemediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other extracellular matrix-related diseases such as cancer and fibrosis.To investigate whether HIF signalling needs to be controlled in growth plate chondrocytes, we conditionally inactivated HIF prolyl hydroxylase 2 (PHD2; Phd2 chonmice), its main negative regulator 7 , resulting in HIF-1α accumulation (Extended Data Fig. 1a-d).This approach caused skeletal dysplasia, characterized by impaired longitudinal bone growth and increased trabecular bone mass (Fig. 1a,b, Extended Data Fig. 1e,f). The growth plate was shorter, but normally organized and, interestingly, the high bone mass was not due to altered bone resorption or formation (Extended Data Fig. 1g-l). Instead, we observed more cartilage remnants in the bony trabeculae, evidenced by more type II collagen (COL2)positive and proteoglycan-rich matrix (Fig. 1c, Extended Data Fig. 1m). The decreased serum CTx-II levels, measuring COL2 degradation, indicated that the cartilage matrix was incompletely resorbed, and the unaltered chondrocyte-to-matrix ratio pointed to a qualitative, rather than quantitative, change in matrix properties (Extended Data Fig. 1j,n). Thus, inactive oxygen sensing in chondrocytes increases trabecular bone mass, caused by abundant cartilage remnants, likely resulting from modifications in the cartilage matrix itself.HIF-1α stabilization in PHD2-deficient chondrocytes resulted, as expected 7,8 , in metabolic reprogramming. Mitochondrial content was reduced, likely because of decreased biogenesis without changing autophagy (Extended D...

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The hydration properties of poly(N-isopropylacrylamide) in aqueous solution were investigated by Fourier transform infrared spectroscopy as a function of high hydrostatic pressure and compared to the thermally induced changes. We show that although both pressure and temperature induce a phase separation the underlying mechanisms are fundamentally different. It is well documented that increasing the temperature above the lower critical solution temperature causes a dehydration of the hydrophilic and hydrophobic moieties. By contrast, high pressure enhances the hydration of the hydrophilic amide group. Moreover, pressure strengthens the weak C−H···O hydrogen bonds between the hydrophobic alkyl groups and water, although a reorganization of the water network around the hydrophobic groups occurs during the phase separation. From this it is concluded that PNiPA remains in a coillike state at high pressure. In addition, we suggest that PNiPA is a good model for the study of the hydration properties of proteins.

High hydrostatic pressure induces conformational changes in proteins ranging from compression of the molecules to loss of native structure. In this tutorial review we describe how the interplay between the volume change and the compressibility leads to pressure-induced unfolding of proteins and dissociation of amyloid fibrils. We also discuss the effect of pressure on protein folding and free energy landscapes. From a molecular viewpoint, pressure effects can be rationalised in terms of packing and hydration of proteins.