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a1-Acid glycoprotein (AGP) is a plasma protein and a member of the acute phase response. AGP is known to bind and carry several biologically active compounds, as well as to down-modulate the immune system activities. In this work, the structure of bovine AGP has been investigated by Fourier-Transform infrared spectroscopy. A model structure has been obtained on the basis of human AGP and refined by molecular dynamics. In spite of the similar structure, bovine AGP shows an unexpectedly higher (w20 � C) thermostability than its human counterpart. Inspection of the model structure has pointed out the presence of 12 ionic bridges and 2 sulphurearomatic interactions, whereas only 6 ionic bridges were detected in human AGP. The high number (9) of glutamic acid residues involved in the ionic interactions might explain the significantly decreased thermostability measured at pH 5.5 (Tm w 71 � C) with respect to pH 7.4 (Tm w 81 � C), whereas thermostability of human AGP was only slightly affected by lowering the pH. As in human AGP and several other lipocalins, a temperature-induced molten globule state has been observed in the denaturation pathway of bovine AGP. 2014 Published by Elsevier Masson SAS.

Human α(1)-acid glycoprotein (AGP) is a positive acute phase plasma protein containing two disulfide bridges. Structural studies have shown that under specific conditions AGP undergoes aggregation. In this study, we analysed the nature of AGP's aggregates formed under reducing and non-reducing conditions at pH 5.5 and at relatively low temperatures. Thioflavin T and Congo red spectroscopic analyses indicated the presence of cross-β structures in both unreduced and reduced AGP aggregates. In these samples amyloid-like fibrils were detected by transmission electron microscopy. The fibrils are branched and bent and present in very large amount in reduced AGP. Kinetics of AGP fibrillation proceeds without a lag phase and the rate constants of cross-β formation are linearly dependent on AGP concentration and result higher under reducing conditions. The data suggest a possible downhill mechanism of polymerization with a first-order monomer concentration dependence. Bioinformatics tools highlighted an extended region that sheathes one side of the molecule containing aggregation-prone regions. Reducing conditions make the extended region less constricted, allowing greater exposure of aggregation-prone regions, thus explaining the higher propensity of AGP to aggregate and fibrillate.

d -serine dehydratase from Saccharomyces cerevisae is a recently discovered dimeric enzyme catalyzing the β-elimination of d -serine to pyruvate and ammonia. The reaction is highly enantioselective and depends on cofactor pyridoxal-5′-phosphate (PLP) and Zn2+. In our work, the aldimine linkage tethering PLP to recombinant, tagged d -serine dehydratase (Dsd) has been reduced by treatment with NaBH4 so as to yield an inactive form of the holoenzyme (DsdR), which was further treated with a protease in order to remove the amino-terminal purification tag. Fourier Transform infrared (FT-IR) spectroscopic analysis revealed that both the reduced form (DsdR) and the reduced/detagged form (DsdRD) maintain the overall secondary structure of Dsd, but featured a significant increased thermal stability. The observed Tm values for DsdR and for DsdRD shifted to 71.5 °C and 73.3 °C, respectively, resulting in nearly 11 °C and 13 °C higher than the one measured for Dsd. Furthermore, the analysis of the FT-IR spectra acquired in the presence of d -serine and l -serine indicates that, though catalytically inert, DsdRD retains the ability to enantioselectively bind its natural substrate. Sequence analysis of d -serine dehydratase and other PLP-dependent enzymes also highlighted critical residues involved in PLP binding. In virtue of its intrinsic properties, DsdRD represents an ideal candidate for the design of novel platforms based on stable, non-consuming binding proteins aimed at measuring d -serine levels in biological fluids.

Human α 1 -acid glycoprotein (AGP) is an acute phase plasma glycoprotein containing two disulfide bridges. As a member of the lipocalin superfamily, it binds and transports several basic and neutral ligands, but a number of other activities have also been described. Thanks to its binding properties, AGP is also a good candidate for the development of biosensors and affinity chromatography media, and in this context detailed structural information is needed. The structural properties of AGP at different p 2 Hs and under reducing conditions were analysed by FT-IR spectroscopy. The obtained data indicate that AGP, when denatured, does not aggregate at neutral or basic p 2 Hs whilst it does at acidic p 2 Hs. Under reducing conditions the protein is remarkably less thermostable than its oxidized counterpart and presents an enhanced tendency to aggregate, even at neutral p 2 H. A heat-induced molten globule-like state (MG) was detected at 55 °C at p 2 H 7.4 and 5.5. At p 2 H 4.5 the MG occurred at 45 °C with an onset of formation at 40 °C. The MG was not observed under reducing conditions. A lower affinity of chlorpromazine and progesterone for the MG formed at p 2 H 4.5 and 40 °C was observed, suggesting that ligand(s) may be released near the negative surfaces of biological membranes. Furthermore, the reduced AGP displays an enhanced affinity for progesterone, indicating the importance of disulfide bonds for the binding capacity of AGP.

d -serine dehydratase (Dsd) from baker’s yeast is a recently discovered enzyme catalyzing the oxidation of d -serine to pyruvate and ammonia. The reaction depends on the cofactors pyridoxal-5′-phosphate (PLP) and Zn2+, featuring a very high selectivity towards the d -enantiomer of the amino acid serine. In humans, altered levels of d -serine in the cerebrospinal fluid (CSF) and blood correlate with the onset and evolution of a number of neurodegenerative diseases. Up to date very little is known on the structure of Dsd. Hence, we have investigated the structure of this enzyme by means of Fourier Transform infrared (FT-IR) spectroscopy and used the structural data derived thereof to validate a homology model of Dsd. In this model, Dsd adopts a fold that is characteristic of type III pyridoxal-dependent enzymes. This consists of an α/β (TIM) barrel and a β-sandwich domain at the N- and C-termini, respectively. Analysis of the Amide I and Amide III infrared bands revealed that the amounts of α (24%), β (29%) and unordered structures (47%) correlate well with those derived from the model (25%, 29% and 46% respectively), suggesting reliability of the latter. In addition, the model of Dsd was further refined by recreating the PLP- and zinc-restored active site based on a PLP- and zinc-dependent bacterial amino acid racemase recently crystallized, allowing us to identify the potential cofactor and metal binding residues of Dsd.

α1-Acid glycoprotein (AGP) is a plasma protein and a member of the acute phase response. AGP is known to bind and carry several biologically active compounds, as well as to down-modulate the immune system activities. In this work, the structure of bovine AGP has been investigated by Fourier-Transform infrared spectroscopy. A model structure has been obtained on the basis of human AGP and refined by molecular dynamics. In spite of the similar structure, bovine AGP shows an unexpectedly higher (∼20 °C) thermostability than its human counterpart. Inspection of the model structure has pointed out the presence of 12 ionic bridges and 2 sulphur-aromatic interactions, whereas only 6 ionic bridges were detected in human AGP. The high number (9) of glutamic acid residues involved in the ionic interactions might explain the significantly decreased thermostability measured at pH 5.5 (Tm ∼ 71 °C) with respect to pH 7.4 (Tm ∼ 81 °C), whereas thermostability of human AGP was only slightly affected by lowering the pH. As in human AGP and several other lipocalins, a temperature-induced molten globule state has been observed in the denaturation pathway of bovine AGP.

Human α(1)-acid glycoprotein (AGP) is an acute phase plasma glycoprotein containing two disulfide bridges. As a member of the lipocalin superfamily, it binds and transports several basic and neutral ligands, but a number of other activities have also been described. Thanks to its binding properties, AGP is also a good candidate for the development of biosensors and affinity chromatography media, and in this context detailed structural information is needed. The structural properties of AGP at different p(2)Hs and under reducing conditions were analysed by FT-IR spectroscopy. The obtained data indicate that AGP, when denatured, does not aggregate at neutral or basic p(2)Hs whilst it does at acidic p(2)Hs. Under reducing conditions the protein is remarkably less thermostable than its oxidized counterpart and presents an enhanced tendency to aggregate, even at neutral p(2)H. A heat-induced molten globule-like state (MG) was detected at 55 °C at p(2)H 7.4 and 5.5. At p(2)H 4.5 the MG occurred at 45 °C with an onset of formation at 40 °C. The MG was not observed under reducing conditions. A lower affinity of chlorpromazine and progesterone for the MG formed at p(2)H 4.5 and 40 °C was observed, suggesting that ligand(s) may be released near the negative surfaces of biological membranes. Furthermore, the reduced AGP displays an enhanced affinity for progesterone, indicating the importance of disulfide bonds for the binding capacity of AGP.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a thermostable tetrameric protein with a molecular mass of 240 kDa which is stable in the presence of detergents and has a maximal activity above 95 degrees C. An understanding of the structure-function relationship of the enzyme under different chemical-physical conditions is of fundamental importance for both theoretical and application purposes. In this paper we report the effect of basic pH values on the structural stability of this enzyme. The structure of the enzyme was studied at pH 10 and in the temperature range 25-97.5 degrees C using circular dichroism, Fourier-transform infrared and fluorescence spectroscopy. The spectroscopic data indicated that the enzyme stability was strongly affected by pH 10 suggesting that the destabilization of the protein structure is correlated with the perturbation of ionic interactions present in the native protein at neutral pHs. These experiments give support to the observation derived from the 3D-structure, that large ion pair networks on the surface stabilize Sulfolobus solfataricus beta-glycosidase.