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The functions of N-acylethanolamines, minor constituents of mammalian cells, are poorly understood. It was suggested that NAEs might have some pharmacological actions and might serve as a cytoprotective response, whether mediated by physical interactions with membranes or enzymes or mediated by activation of cannabinoid receptors. Albumins are identified as the major transport proteins in blood plasma for many compounds including fatty acids, hormones, bilirubin, ions, and many drugs. Moreover, albumin has been used as a model protein in many areas, because of its multifunctional binding properties. Bovine (BSA) and human (HSA) serum albumin are similar in sequence and conformation, but differ for the number of tryptophan residues. This difference can be used to monitor unlike protein domains. Our data suggest that NOEA binds with high affinity to both albumins, modifying their conformational features. In both proteins, NOEA molecules are linked with higher affinity to hydrophobic sites near Trp-214 in HSA or Trp-212 in BSA. Moreover, fluorescence data support the hypothesis of the presence of other NOEA binding sites on BSA, likely affecting Trp-134 environment. The presence of similar binding sites is not measurable on HSA, because it lacks of the second Trp residue.

The thermal unfolding and reversibility of temperature-induced changes in alpha(sub 1)-acid glycoprotein (AGP) is investigated in FT-IR spectroscopy. The resemblance of AGP and odorant-binding protein (OBP) 3D structures confirmed the similarities of data obtained using FT-IR spectroscopy.

Results show that sodium cation induces GTPase activity and the anions modulate the enzyme activity through destabilization of regions of elongation factor 1-alpha from Sulfolobus solfataricus. Infrared analysis indicate particular regions of the polypeptide undergoing structural confromational changes.

TMA-DPH fluorescence decay in human PMN before and after stimulation with FMLP was studied using frequency domain fluorometry. Membrane heterogeneity was assessed by the width of the continuous distributions of lifetime values of Lorentzian shape used to describe the fluorescence decay. In non-stimulated granulocytes TMA-DPH fluorescence decay is characterized by two distributions of lifetime values centered at 6.5 and 1.0 ns and full width at half maximum of 0.3 and 1.2 ns, respectively. Within 15 min after stimulation, the center values of the two distribution components were 5.1 and 0.8 ns and the distribution width was 0.8 and 0.6 ns, respectively. These results indicate changes of membrane domain organization which can be ascribed to compositional changes and redistribution of membrane components.

The identification of the determinants of protein thermal stabilization is often pursued by comparing enzymes from hyperthermophiles with their mesophilic counterparts while direct structural comparisons among proteins and enzymes from hyperthermophiles are rather uncommon. Here, oligomeric β-glycosidases from the hyperthermophilic archaea Sulfolobus solfataricus (Ssβ-gly), Thermosphaera aggregans (Taβ-gly), and Pyrococcus furiosus (Pfβ-gly), have been compared. Studies of FTIR spectroscopy and kinetics of thermal inactivation showed that the three enzymes had similar secondary structure composition, but Ssβ-gly and Taβ-gly (temperatures of melting 98.1 and 98.4°C, respectively) were less stable than Pfβ-gly, which maintained its secondary structure even at 99.5°C. The thermal denaturation of Pfβ-gly, followed in the presence of SDS, suggested that this enzyme is stabilized by hydrophobic interactions. A detailed inspection of the 3D-structures of these enzymes supported the experimental results: Ssβ-gly and Taβ-gly are stabilized by a combination of ion-pairs networks and intrasubunit S-S bridges while the increased stability of Pfβ-gly resides in a more compact protein core. The different strategies of protein stabilization give experimental support to recent theories on thermophilic adaptation and suggest that different stabilization strategies could have been adopted among archaea. Proteins 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The effect of the depletion of calcium on the structure and thermal stability of the d-galactose/d-glucose–binding protein (GGBP) from Escherichia coli was studied by fluorescence spectroscopy and Fourier-transform infrared spectroscopy. The calcium-depleted protein (GGBP-Ca) was also studied in the presence of glucose (GGBP-Ca/Glc). The results show that calcium depletion has a small effect on the secondary structure of GGBP, and, in particular it affects a population of α-helices with a low exposure to solvent. Alternatively, glucose-binding to GGBP-Ca eliminates the effect induced by calcium depletion by restoring a secondary structure similar to that of the native protein. In addition, the infrared and fluorescence data obtained reveal that calcium depletion markedly reduces the thermal stability of GGBP. In particular, the spectroscopic experiments show that the depletion of calcium mainly affects the stability of the C-terminal domain of the protein. However, the binding of glucose restores the thermal stability of GGBP-Ca. The thermostability of GGBP and GGBP-Ca was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results. New insights into the role of calcium in the thermal stability of GGBP contribute to a better understanding of the protein function and constitute important information for the development of biotechnological applications of this protein. Mutations and/or labelling of amino acid residues located in the protein C-terminal domain may affect the stability of the whole protein structure. [ABSTRACT FROM PUBLISHER]

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition. Proteins 2004. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

β-Glycosidase from the extreme thermophilic arch aeon Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95°C. Understanding the structure-activity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus β-glyco-sidase expressed in Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity. [ABSTRACT FROM AUTHOR]

The secondary structure of transglutaminase was investigated by Fourier tranform infrared spectroscopy. Spectra of the protein in both H2O and 2H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide-I band. The quantitative analysis of the amide-I-band components was made by a curve-fitting procedure. The protein was studied in the absence and in the presence of 1mM GTP, 1 mM Ca²+ and 1 mM GTP/1 mM Ca²+. The quantitative analysis of infrared spectra revealed that no remarkable changes, however were observed in the thermal-denaturation behavior of the protein. The protein showed maximum of denaturation at temperature over 50–55°C in the absence or in the presence of 1 mM Ca²+ and over 44–60°C in the presence of 1 mM GTP or 1mM Ca²+/1 mM GTP. The results obtained indicate that GTP induces a stabilization of the tertiary structure of the enzyme, even in the presence of 1 mM Ca²+. The thermal denaturation patterns of the protein suggest the occurrence of Ca²+-dependent aggregation. [ABSTRACT FROM AUTHOR]

CUT-1 from the intestinal parasitic nematode Ascaris lumbricoides is a protein component of the insoluble residue of the cuticle, cuticlin. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The structure and the thermal stability of recombinant CUT-1 from A. lumbricoides (ASCUT-1) were investigated by Fourier-transform infrared (FT-IR) and CD spectroscopy. The data revealed that the secondary structure of the protein at 20 +C, both as insoluble inclusion bodies or in soluble form, contains about 50 % β structure, 14 % α-helix and 25 % turns. A tendency of A. lumbricoides CUT-1 to form aggregates was documented by FT-IR spectroscopy which showed also that the addition of SDS disrupts these interactions. Near-ultraviolet CD spectra confirmed these data and suggested that phenylalanine residues are probably involved in intermolecular hydrophobic interactions responsible for the tendency of the protein to aggregate. Near-ultraviolet spectra showed also that part of the cysteine residues forms disulphide bridges responsible for the tertiary architecture of the protein. Finally, FT-IR and CD data revealed that ASCUT-1 is very stable at high temperatures. This stability and the tendency of ASCUT-1 to form aggregates suggest that these properties may be important for a protein which is a component of a particularly resistant extracellular matrix such as the nematode cuticle. [ABSTRACT FROM AUTHOR]

1-Pyreneisothiocyanate was shown to be an inhibitor of Na+/K+-ATPase. Reverse-phase HPLC and activity studies indicated binding of 1-pyreneisothiocyanate at the H4-H5 loop of the α subunit and competition with the fluorescein 5′-isothiocyanate for the E1ATP site. While fluorescein 5′-isothiocyanate, the fluorescent ATP pseudo-analog, was shown to be immobilized at the E1ATP site, there was no possibility to draw any conclusion about the flexibility of the E1ATP site due to its short lifetime. Employing 1pyreneisothiocyanate as a long-lived fluorophore and a label for the E1ATP site, we found that the ATP-binding site of Na+/K+-ATPase and, in fact, the whole large intracellularly exposed H4-H5 loop of the catalytic α subunit is rigid and rotationally immobilized. This has important consequences for the molecular mechanism of the transport function. [ABSTRACT FROM AUTHOR]