Sheep prion protein synthetic peptide spanning helix 1 and beta-strand 2 (residues 142-166) shows beta-hairpin structure in solution.

According to the "protein only" hypothesis, a conformational conversion of the non-pathogenic "cellular" prion isoform into a pathogenic "scrapie" isoform is the fundamental event in the onset of prion diseases. During this pathogenic conversion, helix H1 and two adjacent surface loops L2 and L3 of the normal prion protein are thought to undergo a conformational transition into an extended beta-like structure, which is prompted by interactions with the pre-existing beta-sheet. To get more insight into the interaction between the helix and one of the beta-strands in the partially unfolded prion protein, the solution structure of a synthetic linear peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. We found that, in contrast to many prion fragments studied earlier, this peptide (i) is highly soluble and does not aggregate up to a millimolar concentration range in aqueous medium and (ii) exhibits an intrinsic propensity to a beta-hairpin like conformation at neutral pH. This beta-propensity can be one of the internal driving forces of the molecular rearrangement responsible for the pathogenic conversion of the prion protein.

Zinc binding to Alzheimer's Abeta(1-16) peptide results in stable soluble complex.

Aggregation of the human amyloid beta-peptide (Abeta) into insoluble plaques is a key event in Alzheimer's disease. Zinc sharply accelerates the Abeta aggregation in vitro, and the Abeta region 6-28 was suggested to be the obligatory zinc binding site. However, time-dependent aggregation of the zinc-bound Abeta species investigated so far prevented their structural analysis. By using CD spectroscopy, we have shown here for the first time that (i) the protected synthetic peptide spanning the fragment 1-16 of Abeta binds specifically zinc with 1:1 and 1:2 stoichiometry under physiologically relevant conditions; (ii) the peptide-zinc complex is soluble and stable for several months; (iii) zinc binding causes a conformational change of the peptide towards a more structured state. These findings suggest the region 1-16 to be the minimal autonomous zinc binding domain of Abeta.

Reversible pressure deformation of a thermophilic cytochrome P450 enzyme (CYP119) and its active-site mutants.

The pressure stability of the thermophilic CYP119 from Sulfolobus solfataricus and its active-site Thr213 and Thr214 mutants was investigated. At 20 degrees C and pH 6.5, the protein undergoes a reversible P450-to-P420 inactivation with a midpoint at 380 MPa and a reaction volume change of -28 mL/mol. The volume of activation of the process was -9.5 mL/mol. The inactivation transition was retarded, and the absolute reaction volume was decreased by increasing temperature or by mutations that decrease the size of the active-site cavity. High pressure affected the tryptophan fluorescence yield, which decreased by about 37% at 480 MPa. The effect was reversible and suggested considerable contraction of the protein. Aerobic decomposition of iron-aryl complexes of the CYP119 T213A mutant under increasing hydrostatic pressure resulted in variation of the N-arylprotoporphyrin-IX regioisomer (N(B):N(A):N(C):N(D)) adduct pattern from 39:47:07:07 at 0.1 MPa to 23:36:14:27 at 400 MPa. Preincubation of the protein at 400 MPa followed by complex formation and decomposition gave the same regioisomer distribution as untreated protein. The results indicate that the protein is reversibly inactivated by pressure, in contrast to the irreversible inactivation of P450(cam) and other P450 enzymes, and that this inactivation process is modulated by changes in the active-site cavity dimensions.

Molecular recognition in the p450cam monooxygenase system: direct monitoring of protein-protein interactions by using optical biosensor.

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins.

Stabilization of P450 2B4 by its association with P450 1A2 revealed by high-pressure spectroscopy.

We studied the effect of intermolecular interactions between cytochromes P450 1A2 (CYP1A2) and 2B4 (CYP2B4) on the barotropic inactivation of the ferrous carbonyl complexes of the hemoproteins. When taken separately, these hemoproteins reveal quite distinct barotropic behavior. While the 2B4(Fe(2+))-CO complex is very sensitive to hydrostatic pressures and undergoes P450 --> P420 transition at rather low pressures (P(1/2) = 297 MPa, DeltaV(0) = -61 ml/mol), the 1A2(Fe(2+))-CO is extremely resistant to barotropic inactivation. Only about 8% of the 1A2 was exposed to pressure-induced P450 --> P420 transition (P(1/2) = 420 MPa, DeltaV(0) = -28 ml/mol). The formation of the mixed oligomers of 2B4 and 1A2 was found to have a dramatic effect on the barotropic behavior of 2B4. In the heterooligomers of 1A2 and 2B4, the 2B4 hemoprotein appears to be largely protected from barotropic inactivation. In 1:1 mixed oligomers no more than 25% of the total P450 content undergoes P450 --> P420 inactivation with the molar reaction volume value (DeltaV(0) = -26 ml/mol) similar to those found for pure 1A2. Moreover, interactions between 1A2 and 2B4 results in a displacement of the Soret band of the ferrous carbonyl complex of CYP2B4 to shorter wavelength (from 451.3 to 448.4 nm) and largely strengthens the dependence of the Soret band wavenumber on hydrostatic pressure below 200 MPa. This effect suggests an important hydration of the CYP2B4 heme moiety in response to the interactions with CYP1A2. We discuss these results in terms of the hypothesis that the heterooligomerization of cytochromes P450 in microsomes plays an important role in the control of the activity and coupling of the microsomal monooxygenase.

Photoacoustic calorimetry of proteins.

We have described two examples of time-resolved photoacoustic calorimetry for the study of heme protein transient intermediates. Before photoacoustic calorimetry, determining thermodynamic information on short-lived intermediates was difficult. Along with being sensitive to enthalpic and volume changes, photoacoustic calorimetry can detect conformational changes in a time-resolved manner. In complex protein systems, the interpretation of the structural origins of a conformational change is sometimes difficult. Site-directed mutagenesis has been used successfully to identify the residues that play important roles in the ligand binding to both Mb and cytochrome P450cam. In both systems the hydration state of salt bridges gave rise to volume changes that were identified through mutagenesis of the residues involved. With its increasing popularity and the power of site-directed mutagenesis, time-resolved photoacoustic calorimetry is fast becoming a technique to probe conformational dynamics in proteins.

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase.

Dilatometry is a sensitive technique for measuring volume changes occurring during a chemical reaction. We applied it to the reduction-oxidation cycle of cytochrome c oxidase, and to the binding of cytochrome c to the oxidase. We measured the volume changes that occur during the interconversion of oxidase intermediates. The numerical values of these volume changes have allowed the construction of a thermodynamic cycle that includes many of the redox intermediates. The system volume for each of the intermediates is different. We suggest that these differences arise by two mechanisms that are not mutually exclusive: intermediates in the catalytic cycle could be hydrated to different extents, and/or small voids in the protein could open and close. Based on our experience with osmotic stress, we believe that at least a portion of the volume changes represent the obligatory movement of solvent into and out of the oxidase during the combined electron and proton transfer process. The volume changes associated with the binding of cytochrome c to cytochrome c oxidase have been studied as a function of the redox state of the two proteins. The volume changes determined by dilatometry are large and negative. The data indicate quite clearly that there are structural alterations in the two proteins that occur on complex formation.

Epitope mapping of cytochrome P450cam (CYP101).

Eighteen linear antigenically active sites were revealed in cytochrome P450 from Pseudomonas putida (P450cam) by hexapeptide scanning. These sites occupy about 31% of the protein sequence. Hexapeptide epitope sequences of P450cam are not found in other cytochromes P450. However, several cytochromes P450 contain shorter fragments of P450cam epitope sequences which may cause weak immune cross-reactions. P450cam antigenic determinants are located generally at the boundaries of secondary structure elements. Mapping of P450cam antigenic determinants on the three-dimensional structure of this protein reveals 14 highly water-accessible antigenic sites and only 1 site (No. 322-327, QMLSGL) which is inaccessible to water. Several functionally important sites and amino acid residues of P450cam are localized within revealed linear epitopes or very close to them. These sites include substrate-binding regions, residues responsible for the putidaredoxin interaction (Arg72, Arg112, Lys314, and Arg364), heme binding (Gln108, Arg112, Asp297, Arg299, and Cys357), and proton translocation (Lys178, Arg186, and Glu366).

Mobility of norbornane-type substrates and water accessibility in cytochrome P-450cam.

The behaviour of norbornane-type substrates bound to oxidised cytochrome P-450cam (CYP 101) in 60% (w/w) glycerol-containing phosphate buffer was investigated using electronic absorption spectroscopy. The high-pressure dependence study revealed that the value of the spin-state reaction-volume change decreased from -70 to -22.8 cm3/mol with decreasing high-spin state content from 99 to 63%. Simultaneously, the values for the enthalpy and entropy determined from the low-temperature dependence of the spin-state transition decreased from 73.7 to 24.3 kJ/mol and from 310.4 to 88.9 J/mol K, respectively. Under our experimental conditions the pH-value of the buffer remained at low temperatures and high pressures in the range of pH 7-8, in which no pH-value-induced spin-state conversion occurred. Therefore, the secondary effect of the temperature and pressure-induced pH change can be disregarded as being responsible for the observed spin-state transition effects. Substrate dissociation constants were determined. From the temperature-jump experiments (297 K to 180 K) we found a higher mobility in the active site for the substrates in the sequence (1R)-camphor, (1S)-camphor, camphane, (1R)- and (1S)-camphorquinone, norcamphor, and norbornane. Our findings can be explained by the incomplete fit of the methyl groups of the norbornane-type substrate to the protein, in particular to the I-helix, predominantly determining the substrate mobility and water accessibility to the protein.

Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes.

An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described. The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate. This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes. These parameters are discussed in comparison with the values reported earlier by Ristau et al. [(1979) Acta Biol. Med. Ger. 38, 177-185] for rabbit liver cytochrome P-450 2B4 in solution with benzphetamine as a substrate. Our analysis shows the substrate-free states of 2B4 and 3A4 to be very similar. However, substrate binding seems to perturb haem-protein interactions in 3A4 in contrast with 2B4, where the effect of substrate binding on the thermodynamic parameters of spin transitions was insignificant. The implication of the results for the mechanism of substrate-induced spin shift is discussed.

Structural changes in cytochrome P-450cam effected by the binding of the enantiomers (1R)-camphor and (1S)-camphor.

A comparative study of the enantiomeric substrate [(1R)-camphor- and (1S)-camphor)-bound cytochrome P-450cam concerns the spin-state equilibrium, substrate dissociation, the thermal unfolding of the protein structure, and the subconformer equilibria observed in the infrared spectra of the carbon monoxide (CO) complex of cytochrome P-450cam. The behavior of the different conformational equilibria in dependence on temperature, pressure, pH-value, cosolvent, and cation binding led us to suggest that (1S)-camphor is more loosely and less optimally bound in the heme pocket, which facilitates the access of solvent molecules into the heme-iron environment. The spin reaction volume difference measured using the high pressure technique is smaller by 16 +/- 9 cm3/mol for (1S)-camphor-bound P-450cam compared to the (1R)-camphor-bound P-450cam, which might indicate a higher water content in the protein and in the heme environment in the (1S)-camphor complex. The half-transition temperature of the thermal unfolding of 53.8 degrees C for the (1S)-camphor-bound oxidized cytochrome P-450cam is one degree lower than the value for the (1R)-camphor-bound protein (54.8 degrees C). In the reduced, CO-bound form of cytochrome P-450cam at 290 K the (1S)-camphor complex reveals another CO stretch vibration population distribution with slightly higher frequencies [1940.2 cm-1 (major band) and 1946.3 cm-1 (minor band)] compared to the (1R)-camphor complex [1939.7 cm-1 (major band) and 1930 cm-1 (minor band)]. A loosening of the contact between the iron-bound CO ligand and amino acids of the I-helix, probably induced by compensating effects of the increased water content, is suggested. Assuming the carbon monoxide complex as a model for the dioxygen complex, the more loosened binding of (1S)-camphor, therefore the increased water accessibility, and the weaker contact of the iron ligand to the I-helix might explain the higher amount of uncoupling of the cytochrome P-450 reaction cycle compared to that when (1R)-camphor is used as substrate.

Probing the heme iron coordination structure of pressure-induced cytochrome P420cam.

Cytochrome P450cam was subjected to high pressures of 2.2 kbar, converting the enzyme to its inactive form P420cam. The resultant protein was characterized by electron paramagnetic resonance, magnetic circular dichroism, circular dichroism, and electronic absorption spectroscopy. A range of exogenous ligands has been employed to probe the coordination structure of P420cam. The results suggest that conversion to P420cam involves a conformational change which restricts the substrate binding site and/or alters the ligand access channel. The reduction potential of P420cam is essentially the same in the presence or absence of camphor (-211 +/- 10 and -210 +/- 15 mV, respectively). Thus, the well-documented thermodynamic regulation of enzymatic activity for P450cam in which the reduction potential is coupled to camphor binding is not found with P420cam. Further, cyanide binds more tightly to P420cam (Kd = 1.1 +/- 0.1 mM) than to P450cam (Kd = 4.6 +/- 0.2 mM), reflecting a weakened iron-sulfur ligation. Spectral evidence reported herein for P420cam as well as results from a parallel investigation of the spectroscopically related inactive form of chloroperoxidase lead to the conclusion that a sulfur-derived proximal ligand is coordinated to the heme of ferric cytochrome P420cam.

High-pressure-induced transitions in microsomal cytochrome P450 2B4 in solution: evidence for conformational inhomogeneity in the oligomers.

Pressure-induced changes in ferric P450 2B4 (LM2) were studied as a function of benzphetamine concentration (0.05 divided by 2 mM) and state of aggregation of the hemoprotein in solution. Application of factor analysis to the spectral changes in the Soret region allowed us to resolve two particular pressure-induced processes in 2B4 oligomers. The first process was identified as the conversion of the low-spin P450 into the P420 state. At 25 degrees C it was followed by decay (bleaching) of about 50% of the newly formed P420. The second process was a pressure-induced high- to low-spin shift. Both transitions were reversible, except the hemoprotein bleaching. The amplitude of the P450-->P420 transition accounted for 67 +/- 5% of the total hemoprotein content. Furthermore, the fraction of the hemoprotein exposed to spin equilibrium was not affected by the P450-->P420 conversion and was estimated to be only about 31 +/- 5% of the total hemoprotein content. After the dissociation of the oligomers by 0.2% Triton N-101, the inhomogeneity vanished: 95% of the monomers were involved in the P450-->P420 transition (delta V degrees = -86 ml/mol) followed by intense bleaching of the hemoprotein. This agrees with our earlier observations on the reduced carbonyl complex of P450 2B4 and suggests some conformational difference between subunits in P450 LM2 oligomers. The parameters of the P450-->P420 conversion (delta V degrees = -32 ml/mol, P1/2 = 1560 bar) show no dependency on the substrate concentration. Analysis of the pressure-induced spin shift versus benzphetamine concentration shows this transition to be caused mainly by changes in the spin equilibrium of both substrate-bound (delta V degrees = -49 ml/mol) and substrate-free (delta V degrees = -21 ml/mol) hemoprotein, whereas the substrate binding step itself has a very weak pressure dependency (delta V degrees = -8 ml/mol).

Antagonistic effects of hydrostatic pressure and osmotic pressure on cytochrome P-450cam spin transition.

The combined effects of hydrostatic pressure and osmotic pressure, generated by polyols, on the spin equilibrium of fenchone-bound cytochrome P-450cam were investigated. Hydrostatic pressure indices a high spin to low spin transition, whereas polyols induce the reversed reaction. Of the four solutes used, glycerol, glucose, stachyose, and sucrose, only the last two would act on the spin transition by osmotic stress. The spin volume changes measured by both techniques are different, 29 and -350 ml/mol for hydrostatic pressure and osmotic pressure, respectively. It suggests that even if the two are perturbing water molecules, different properties are probed. From the volume change induced by osmotic stress, 19 water molecules are deduced that would be implicated in the spin transition of the fenchone-bound protein. This result suggests that water molecules other than the well defined ones located in the active site play a key role in modulating the spin equilibrium of cytochrome P-450cam.

Second derivative spectroscopy of enolase at high hydrostatic pressure: an approach to the study of macromolecular interactions.

Second derivative spectroscopy in the ultraviolet region of proteins has been used to study the polarity of the regions surrounding tyrosine residues. We show here that it can also be a tool to study the degree to which proteins associate and that it can be effectively combined with hydrostatic pressure in order to evaluate equilibrium dissociation constants and reaction volumes. Hydrostatic pressure causes yeast enolase to dissociate. Clear changes in the second derivative spectra of enolase were observed as pressure was increased. At enolase concentrations of about 20 microM, the midpoint of the transition is about 1800 bar. All aspects of the transition are reversible up to 2700 bar. It is likely that the transition observed is the result of enolase dimers dissociating into monomers. The second derivative spectra indicate that one or more tyrosine residues is in an unusually polar environment in the dimer, an environment that is less polar in the monomer. Three tyrosines (6, 11, 130) are near the dimer interface. Tyrosines 6 and 11 are pointing into the water-filled crevice between the subunits and are close to several immobilized waters. All three are close to a network of intersubunit salt bridges and hydrogen bonds. We believe that the average tyrosine polarity in the dimer reflects the exposure of these tyrosines to immobilized water and the fixed dipole of the salt bridge. The water in the crevice between the subunits should be more mobile in the monomer; the salt bridge does not exist in the monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

High-pressure effects on beta-lactoglobulin interactions with ligands studied by fluorescence.

The effects of pressure (0.1 MPa to 400 MPa) on intrinsic fluorescence of beta-lactoglobulin and on its binding of retinol and cis-parinaric acid have been studied at neutral and acid pHs. In neutral pH, fluorescence emission spectra of beta-lactoglobulin tryptophanes are characterized by an irreversible 14 nm red-shift indicating pressure-induced folding changes. The intensity of the fluorescence of retinol in beta-lactoglobulin-retinol complex is enhanced by a pressure increase up to 150 MPa. It decreases at higher pressures and disappears altogether at 300 MPa. beta-Lactoglobulin-retinol complex does not reassociate after decompression at neutral pH. At acid pH condition, the fluorescence quenching by pressure of beta-lactoglobulin tryptophans is coupled with a 2 nm spectral shift and is fully reversible demonstrating almost complete restoration of globulin folding. The evolution of retinol fluorescence in beta-lactoglobulin-retinol complex is also entirely reversible between 0.1 MPa and 400 MPa and the complex never dissociates in the studied pressure range. beta-lactoglobulin-cis-parinaric acid complexes at neutral and acid pH values dissociate irreversibly at 200 MPa and 350 MPa, respectively.

Effects of monovalent cations on cytochrome P-450 camphor. Evidence for preferential binding of potassium.

Binding of monovalent cations of increasing ionic radius to ferric cytochrome P-450cam was measured. Potassium has the highest affinity for the cation binding site observed in the X-ray crystallographic structure with Kdcat = 12 mM, compared with the smaller cation lithium, (Kdcat = 37 mM) and the larger cation cesium (Kd cat = 20 mM). Coupling between cation binding and camphor binding is established by the observation of a linear relationship between the corresponding binding free energies. Potassium binding favours a conformational change of tyrosine 96 which increases the affinity of the protein for camphor and fully dehydrates the active site.

Responses of two protein-protein complexes to solvent stress: does water play a role at the interface?

We have analyzed the stability of the cytochrome c-cytochrome b5 and cytochrome c-cytochrome c oxidase complexes as a function of solvent stress. High concentrations of glycerol were used to displace the two equilibria. Glycerol promotes complex formation between cytochrome c and cytochrome b5 but inhibits that between cytochrome c and cytochrome c oxidase. The results with cytochrome b5 and cytochrome c were expected; the association of this complex is largely entropy driven. Our interpretation is that the cytochrome c-cytochrome b5 complex excludes water. The results with the cytochrome c oxidase and cytochrome c couple were not expected. We interpret them to mean that either glycerol is binding to the oxidase, thereby displacing the cytochrome c, or that water is required at this protein-protein interface. A requirement for substantial quantities of water at the interface of some protein complexes is logical but has been reported only once.

Substrate analogue induced changes of the CO-stretching mode in the cytochrome P450cam-carbon monoxide complex.

The CO-stretching mode of the carbon monoxide ligand in reduced cytochrome P450cam, in the absence or presence of camphor and in the presence of nine different camphor analogues, was measured at room temperature using Fourier transform infrared spectroscopy. Substrate-free cytochrome P450cam--CO reveals a broad, slightly structured band resulting from an overlap of several stretching mode signals. The multitude of the signals indicates that cytochrome P450 exists in a dynamic equilibrium of several conformational substates. Binding of camphor or camphor analogues strongly influences this equilibrium. For substrate analogues which are not able to form a hydrogen bond to the hydroxyl group of tyrosine 96, the CO-stretching band is rather broad and asymmetric. In contrast, substrate analogues with one quinone group which form a hydrogen bond to the Tyr96 OH induce a shift and a sharpening of the CO-stretching mode band. For substrate analogues with two hetero groups, the infrared spectrum is slightly asymmetric or a minor band appears. Sterical hindrance, substrate mobility, and protein flexibility finally determine the position and width of the CO-stretching mode signals.

A critical role of protein-bound water in the catalytic cycle of cytochrome P-450 camphor.

The rates of NADH oxidation during the hydroxylation of camphor by cytochrome P-450cam were followed in the presence of co-solvents used to increase the osmotic pressure surrounding the protein-bound water. As a result, the measured Vmax decreases independently of the perturbant tested. Roughly 28 molecules of water, involved during the catalytic cycle, are deduced from the variation of Vmax as a function of osmotic pressure. These molecules, in part, could be those present in the cytochrome P-450cam-putidaredoxin interface.