A common mechanism of conformational changes and pathological aggregation of proteins associated with amyloid diseases remains to be proven. High pressure is emerging as a new strategy for studying aspects of amyloid formation. Pressure provides a convenient means to populate and characterize partially folded states, which are thought to have a key role in assembly processes of proteins into amyloid fibrils.
View Article and Find Full Text PDFBiochim Biophys Acta
March 2006
Sulfolobus solfataricus carboxypeptidase, (CPSso), is a heat- and pressure-resistant zinc-metalloprotease. Thanks to its properties, it is an ideal tool for investigating the role of non-covalent interactions in substrate binding. It has a broad substrate specificity as it can cleave any N-blocked amino acid (except for N-blocked proline).
View Article and Find Full Text PDFWithout being comprehensive in this mini-review, I will address perspectives, some speculative, for the development and use of high pressure to explore biochemical phenomena. This will be illustrated with several examples.
View Article and Find Full Text PDFBiochim Biophys Acta
August 2005
The pressure dependence of enzyme catalytic parameters allows volume changes associated with substrate binding and activation volumes for the chemical steps to be determined. Because catalytic constants are composite parameters, elementary volume change contributions can be calculated from the pressure differentiation of kinetic constants. Linear and non-linear pressure-dependence of single-step enzyme reactions and steady-state catalytic parameters can be observed.
View Article and Find Full Text PDFThe native conformation of host-encoded cellular prion protein (PrP(C)) is metastable. As a result of a post-translational event, PrP(C) can convert to the scrapie form (PrP(Sc)), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown.
View Article and Find Full Text PDFPressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.
View Article and Find Full Text PDFThe effects of hydrostatic pressure on yeast enolase have been studied in the presence of 1 mm Mn(2+). When compared with apo-enolase, and Mg-enolase, the Mn-enzyme differs from the others in three ways. Exposure to hydrostatic pressure does not inactivate the enzyme.
View Article and Find Full Text PDFOur understanding of conformational conversion of proteins in diseases is essential for any diagnostic and therapeutic approach. Although not fully understood, misfolding of the prion protein (PrP) is implicated in the pathogenesis of prion diseases. Despite several efforts to produce the pathologically misfolded conformation in vitro from a recombinant PrP, no positive result has yet been obtained.
View Article and Find Full Text PDFThe rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown.
View Article and Find Full Text PDFThe heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75 degrees C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety.
View Article and Find Full Text PDFThe kinetics of formation and transformation of oxygen complexes of two heme-thiolate proteins (the F393H mutant of cytochrome P450 BM3 and the oxygenase domain of endothelial nitric oxide synthase, eNOS) were studied under high pressure. For BM3, oxygen-binding characteristics (rate and activation volume) matched those measured for CO-binding. In contrast, pressure revealed a different CO- and oxygen-binding mechanism for eNOS, suggesting that it is hazardous to take CO-binding as a model for oxygen-binding.
View Article and Find Full Text PDFSheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai)
July 2003
The unfolding of 23kD (P23k) protein isolated from spinach photosystem II particle was studied by high pressure and fluorescence spectroscopy. The thermal equilibrium study indicated that the protein could be totally unfolded by 180 or 160 MPa at 20 degrees C and 3 degrees C, respectively. The standard free energy and standard volume change of the protein for unfolding at 20 degrees C is 23.
View Article and Find Full Text PDFBiochem Biophys Res Commun
June 2003
The cellular prion protein (PrP(c)) forms complexes with plasminogen. Here, we show that the PrP(c) in this complex is cleaved to yield fragments of PrP(c). The cleavage is accelerated by plasmin but does not appear to be dependent on it.
View Article and Find Full Text PDFWe have studied the reaction native left arrow over right arrow denatured for the 33-kDa protein isolated from photosystem II. Sucrose and glycerol have profound effects on pressure-induced unfolding. The additives shift the equilibrium to the left; they also cause a significant decrease in the standard volume change (DeltaV).
View Article and Find Full Text PDFOverproduction and purification of the prion protein is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. We have developed a method for the effective cloning, overexpression in Escherichia coli and purification to homogeneity of Syrian golden hamster prion protein (SHaPrP(90-231)). A high level of overexpression, resulting in the formation of inclusion bodies, was obtained under the control of the T7-inducible promoter of the pET15b plasmid.
View Article and Find Full Text PDFAt high temperature, recombinant hamster prion protein (SHaPrP(90-231)) undergoes aggregation and changes from a predominantly alpha-helical to beta-sheet conformation. We then applied high pressure (200 MPa) to the beta-sheet-rich conformation. The aggregation was reversed, and the original tertiary and secondary structures were recovered at ambient pressure, after pressure release.
View Article and Find Full Text PDFThe process of pressure-induced modification of horse liver alcohol dehydrogenase (HLADH) was followed by measuring in situ catalytic activity (up to 250 MPa), intrinsic fluorescence (0.1-600 MPa) and modifications of FTIR spectra (up to 1000 MPa). The tryptophan fluorescence measurements and the kinetic data indicated that the pressure-induced denaturation of HLADH was a process involving several transitions and that the observed transient states have characteristic properties of molten globules.
View Article and Find Full Text PDFJ Biomed Biotechnol
January 2001
High-pressure (HP) biotechnology is an emerging technique initially applied for food processing and more recently in pharmaceutical and medical sciences. Pressure can stabilize enzymes and modulate both their activity and specificity. HP engineering of proteins may be used for enzyme-catalyzed synthesis of fine chemicals, pharmaceuticals, and production of modified proteins of medical or pharmaceutical interest.
View Article and Find Full Text PDFSheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai)
January 1999
The goal of this mini review is to explain how hydrostatic pressure acts on bio-macromolecules(mainly on protein), giving some basic knowledge. Moreover, it will be shown that high pressure is a powerful tool both for the study of bio-macromolecules such as protein conformation, protein-protein (or protein-nucleic acid, or protein-ligands) interactions and the modulation of enzymatic activity etc. and for study of biotechnological application.
View Article and Find Full Text PDFAn abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues.
View Article and Find Full Text PDFHydrolysis of the neutral substrate N-methylindoxyl acetate (NMIA) by wild-type human butyrylcholinesterase (BuChE) and peripheral site mutants (D70G, Y332A, D70G/Y332A) was found to follow the Michaelis-Menten kinetics. K(m) was 0.14 mM for wild-type, and 0.
View Article and Find Full Text PDFBiochim Biophys Acta
March 2002
Through some typical examples, the high pressure static fluorescence method is described. The potentiality of the intrinsic and extrinsic fluorescence probes are analyzed for structural characterizations. Special attention is given to the use of fluorescence to understand the behavior of enzymatic reactions under high pressure.
View Article and Find Full Text PDFHigh hydrostatic pressure affects proteins, changing their intra- or intermolecular interactions, conformation and solvation. How to detect these changes? In this paper, via some selected examples, we show the potentiality (but also the limits) of the ultraviolet derivative spectroscopy specially adapted to high pressure experiments.
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