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Biomedical subjects

Gaston Hui Bon Hoa

Publications and source records attributed to Gaston Hui Bon Hoa.

10 recordsLinked to original sources
PubMed2006

Reversible hexacoordination of alpha-hemoglobin-stabilizing protein (AHSP)/alpha-hemoglobin Versus pressure. Evidence for protection of the alpha-chains by their chaperone.

Using high hydrostatic pressure or hydrogen peroxide as perturbing agents, we demonstrate a protective effect of the chaperone AHSP for the alpha-chains of Hb. High pressure induces an irreversible aggregation of the ferrous deoxy alpha-chains, whereas the AHSP/alpha-Hb complex shows reversible hexacoordination of the alpha-Hb without protein aggregation. Upon pressure release, the relaxation kinetics of the transition from the hexacoordinated to pentacoordinated form of alpha-Hb in the presence of AHSP exhibit a biphasic shape. High pressure did not induce dissociation of alpha-Hb from its chaperone, as evidenced by the ligand binding kinetics that show a unique rate for the AHSP/alpha-Hb complex. For both free alpha-Hb and the AHSP/alpha-Hb complex, the bimolecular rate constant of CO binding (k(CO)(on)) versus pressure exhibits a bell shape, attributed to the transition of the rate-determining step from the chemical barrier to the migration of CO within the protein matrix. These results reveal a plasticity of the alpha-Hb active site in the presence of the chaperone and indicate that the AHSP was still active at 300 MPa. The ferric state of the AHSP/alpha-Hb complex shows hexacoordination even at atmospheric pressures, indicating a His-Fe-His binding scheme as previously observed in neuroglobin and cytoglobin. The reaction with hydrogen peroxide of ferric alpha-Hb within the complex also demonstrates a protection against aggregation.

Blood Proteins↗
PubMed2005

High pressure enhances hexacoordination in neuroglobin and other globins.

The techniques of high applied pressure and flash photolysis have been combined to study ligand rebinding to neuroglobin (Ngb) and tomato Hb, globins that may display a His-Fe-His hexacoordination in the absence of external ligands. High pressure induces a moderate decrease in the His association rate and a large decrease in His dissociation rate, thus leading to an enhancement of the overall His affinity. The overall structural difference between penta- and hexacoordinated globins may be rather small and can be overcome by external modifications such as high pressure. Over the pressure range 0.1-700 MPa (7 kbar), the globins may show a loss of over a factor of 100 in the amplitude of the bimolecular rebinding phase after photodissociation. The kinetic data show that pressure induces a moderate increase of the rate for ligand binding from the correlated pair state (just after photodissociation) and a large (factor of 1000) decrease in rate for migration through the protein. The effect on the ligand migration phase was similar for both the external ligands (such as oxygen) as for the internal (histidine) ligand, suggesting the dominant role of protein fluctuations, rather than specific chemical barriers. Thus high pressure efficiently closes the protein migration channels; however, contrary to the effect of high viscosity, high pressure induces a greater decrease in rate for ligand migration toward the exterior (heme to the solvent) versus inward migration, as if the presence of the ligand itself induces an additional steric constraint.

Animals↗
PubMed2004

Possible role of region 152-156 in the structural duality of a peptide fragment from sheep prion protein.

The conformational conversion of the nonpathogenic "cellular" prion isoform into a pathogenic "scrapie" protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a beta-like structure. A peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a beta-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152-156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein.

Animals↗
PubMed2004

Pressure dependence of the dual luminescence of twisting molecules. The case of substituted 2,3-naphthalimides.

The effect of high pressure (up to 5 kbar) has been studied for triacetin solutions of 2-phenyl-2,3-dihydro-1H-benzo[f]isoindole-1,3dione 1 (N-phenyl-2,3-naphthalimide) and its 3-fluorophenyl- (2), 4-carbethoxyphenyl(4) and 4-methoxyphenyl (5) derivatives which all show dual fluorescence. When the N-phenyl group is unsubstituted (compound 1) or substituted with electron-attracting groups (2 and 4), the increase of pressure over the solution decreases slightly the emission at the long-wavelengths (LW) and increases dramatically the intensity of the short-wavelength (SW) fluorescence. Plotting the logarithm of the SW/LW fluorescence quantum yield ratio for compounds, 1,2 and 4 versus the logarithm of the viscosity of the medium shows a substantial increase of this ratio which corresponds mainly to the increase of the SW emission intensity, the effect on the LW emission being only moderate. As the pressure is increased, the rotation of the N-phenyl group of compound 1 is progressively hindered and the prevailing emission comes from a state which has the same geometry as the ground state (in which the planes of the two moieties of the molecule form an angle close to 60 degree). The effect is different when an electron-donating methoxy group is attached in the para position to the N-phenyl ring, compound 5, as mainly the LW fluorescence intensity increases with pressure. For this molecule which has an electron-donating p-substituent on the N-phenyl ring, the two moieties of the ground state molecule have a more planar geometry (43 degree angle) and the LW fluorescence appears to originate from an intramolecular charge transfer state the fluorescence of which increases with pressure. A three-level reaction scheme is proposed to account for the observed kinetics. In all cases, the viscosity of the medium is found to be the main factor which induces the changes in the fluorescence spectra, and the deceleration of the non-radiative deactivation from the SW* excited state is responsible for these modifications whether a reversible process between the two emitting SW* and LW* states is observed (as for compounds 2 and 4) or not (as for compound 1).

Journal Article↗
PubMed2003

Compressibility and uncoupling of cytochrome P450cam: high pressure FTIR and activity studies.

The effect of the hydrostatic pressure on the CO ligand stretch vibration in cytochrome P450cam-CO bound with various substrates is studied by FTIR. The vibration frequency is linearily shifted to lower values with increasing pressure. The slope of the shift gives the isothermal compressibility of the heme pocket and is found to be related to the high-spin state content in an opposite direction to that previously observed from the pressure-induced shift of the Soret band. This opposite behaviour is explained by the dual effect of heme pocket water molecules both on the CO ligand and on electrostatic potentials produced by the protein at the distal side. The latter effect disturbs ligand-distal side contacts which are needed for a specific proton transfer in oxygen activation when dioxygen is the ligand. Their loss results in uncoupled H(2)O(2) formation.

Camphor 5-Monooxygenase↗
PubMed2003

Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy.

We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids.

Bromocriptine↗
PubMed2002

Specific and non-specific effects of potassium cations on substrate-protein interactions in cytochromes P450cam and P450lin.

Substrate binding to cytochrome P450cam is generally considered to be a two-step process. The first step corresponds to the entrance of the substrate, camphor, into the heme pocket. The second step corresponds to a spin transition (low spin-->high spin) of the iron in the protein-substrate complex. This spin transition is related to the mobility of the substrate inside the active site [Biochim Biophys Acta 1338 (1997) 77]. Potassium cations (K(+)) have a specific effect on the spin equilibrium. This is generally attributed to the K(+) ion-induced conformational change of tyrosine 96, the hydroxyl group of which is hydrogen bonded to the keto group of camphor and results in optimum substrate orientation and reduced mobility of this substrate in the active site. In the present paper, we show that K(+) not only affects the substrate-Tyr 96 couple, but acts more globally since K(+) effects are also observed in the Tyr96Phe mutant as well as in complexes with camphor-analogues. Large compounds, that fit well in the heme pocket and bind with higher affinity than camphor, display high spin contents that are less dependent on the presence of K(+). In contrast, K(+) has a significant effect on the high spin content of substrate-cytochrome P450cam complexes with looser interactions. We conclude that large compounds with higher affinities than camphor have more van der Waals contacts with the active site residues. Their mobilities are then reduced and less dependent on the presence of K(+). In this study, we also explored, for comparison, the K(+) effect on the spin transition state of another member of the P450 superfamily, cytochrome P450lin. This effect is not as strong as those observed for cytochrome P450cam. Even though the spin equilibrium does not change dramatically in the presence of K(+) or Na(+), the value of the dissociation constant (K(d)) for linalool binding is significantly affected by ionic strength. Analysis of the thermodynamic parameters for the linalool binding strongly suggests that, similarly to our previous finding for cytochrome P450cam, electrostatic gates participate in the control of substrate access.

Amino Acid Sequence↗
PubMed2002

High pressure, a tool for exploring heme protein active sites.

High pressure is an interesting and suitable parameter in the study of the dynamics and stability of proteins. The effects of pressure on proteins delineates its volumic (deltaV degrees ) and energetic (deltaG degrees ) parameters. An enormous amount of effort has been invested by several laboratories in developing basic theory and high pressure techniques that allow the determination of barotropic parameters. Cytochrome P450s, one of the largest super families of heme proteins, are good models for high pressure studies. Two distinct pressure-induced spin transitions of the heme iron in the active site and a P450 to P420 inactivation process have been characterized. The obtained reaction volumes of these two processes for a series of analog-bound cytochrome P450s are compared. We have shown that both the spin volume and the inactivation volume are dependent on the substrate analogs which are known to modulate the polarity and hydration of the heme pocket. Several linear correlations were found between these reaction volumes and the physico-chemical properties of the heme protein such as the polarity-induced exposure of tyrosines, the hydration of the cytochrome CYP101 heme pocket, and the mobility and binding of the substrates indicate that they constitute the main contribution to the complex thermodynamic reaction volume parameters. This interpretation allows us to conclude that cytochrome CYP101, CYP2B4 and CYP102 possess a similar mechanism of substrate binding. Interestingly the barotropic behaviors of monomeric cytochrome P450s are quite different from those of oligomeric and hetorooligomeric cytochrome P450s. The interactions of heterooligomeric subunits influence the stability of individual cytochrome P450s and the asymmetric organization of subunits which can control and modulate the activity and the recognition with NADPH-cytochrome P450 reductase.

Binding Sites↗