Biomedical subjects

Reinhard Lange

Publications and source records attributed to Reinhard Lange.

At least 19 recordsLinked to original sources

PubMed2006

The contribution of the residues from the main hydrophobic core of ribonuclease A to its pressure-folding transition state.

The role of hydrophobic interactions established by the residues that belong to the main hydrophobic core of ribonuclease A in its pressure-folding transition state was investigated using the Phi-value method. The folding kinetics was studied using pressure-jump techniques both in the pressurization and depressurization directions. The ratio between the folding activation volume and the reaction volume (beta p-value), which is an index of the compactness or degree of solvation of the transition state, was calculated. All the positions analyzed presented fractional Phi f-values, and the lowest were those corresponding to the most critical positions for the ribonuclease A stability. The structure of the transition state of the hydrophobic core of ribonuclease A, from the point of view of formed interactions, is a relatively, uniformly expanded form of the folded structure with a mean Phi f-value of 0.43. This places it halfway between the folded and unfolded states. On the other hand, for the variants, the average of beta p-values is 0.4, suggesting a transition state that is 40% native-like. Altogether the results suggest that the pressure-folding transition state of ribonuclease A looks like a collapsed globule with some secondary structure and a weakened hydrophobic core. A good correlation was found between the Phi f-values and the Deltabeta p-values. Although the nature of the transition state inferred from pressure-induced folding studies and the results of the protein engineering method have been reported to be consistent for other proteins, to the best of our knowledge this is the first direct comparison using a set of mutants.

Amino Acid Sequence↗

PubMed2006

The use of pressure-jump relaxation kinetics to study protein folding landscapes.

Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refolding. These processes can be initiated by upward and downward pressure-jumps of amplitudes of a few 10 to 100 MPa, with a dead-time on the order of milliseconds. In many cases, the relaxation times can be easily determined when the pressure cell is connected to a spectroscopic detection device, such as a spectrofluorimeter. Adiabatic heating or cooling can be limited by small pressure-jump amplitudes and a special design of the sample cell. Here, we discuss the application of this method to four proteins: 33-kDa and 23-kDa proteins from photo-system II, a variant of the green fluorescent protein, and a fluorescent variant of ribonuclease A. The thermodynamically predicted equivalency of upward and downward pressure-jump induced protein relaxation kinetics for typical two-state folders was observed for the 33-kDa protein, only. In contrast, the three other proteins showed significantly different kinetics for pressure-jumps in opposite directions. These results cannot be explained by sequential reaction schemes. Instead, they are in line with a more complex free energy landscape involving multiple pathways.

Animals↗

PubMed2006

Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions Sulfolobus solfataricus carboxypeptidase as a model enzyme.

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). Its catalytic and kinetic mechanisms are well understood, and the hydrolytic reaction is easily detectable spectrophotometrically. Here, we report investigations on the pressure- and temperature-dependence of the kinetic parameters (turnover number and Michaelis constant) of CPSso using several benzoyl- and 3-(2-furyl)acryloyl-amino acids as substrates. This approach enabled us to study these parameters in terms of individual rate constants and establish that the release of the free amino acid is the rate-limiting step, making it possible to dissect the individual non-covalent interactions participating in substrate binding. In keeping with molecular docking experiments performed on the 3D model of CPSso available to date, our results show that both hydrophobic and energetic interactions (i.e., stacking and van der Waals) are mainly involved, but their contribution varies strongly, probably due to changes in the conformational state of the enzyme.

Archaeal Proteins↗

PubMed2006

Pressure- and temperature-induced unfolding studies: thermodynamics of core hydrophobicity and packing of ribonuclease A.

In this work we demonstrate that heat and pressure induce only slightly different energetic changes in the unfolded state of RNase A. Using pressure and temperature as denaturants on a significant number of variants, and by determining the free energy of unfolding at different temperatures, we estimated the stability of variants unable to complete the unfolding transition owing to the experimental conditions required for pressure experiments. The overall set of results allowed us to map the contributions to stability of the hydrophobic core residues of RNase A, with the positions most critical for stability being V54, V57, I106 and V108. We also show that the stability differences can be attributed to both hydrophobic interactions and packing density with an equivalent energetic magnitude. The main hydrophobic core of RNase A is tightly packed, as shown by the small-to-large and isosteric substitutions. In addition, we found that large changes in the number of methylene groups have non-additive positive stability interaction energies that are consistent with exquisite tight core packing and rearrangements of van der Waals' interactions in the protein interior, even after drastic deleterious substitutions.

Enzyme Stability↗

PubMed2006

High pressure modulates amyloid formation.

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. High pressure can also be used to dissociate aggregates and amyloid fibrils or on the opposite to generate such species.

Amyloid↗

PubMed2005

High-pressure studies of the reaction mechanism of nitric-oxide synthase.

Nitric-oxide synthase (NOS) generates nitric oxide from l-arginine in two reaction cycles with N(omega)-hydroxy-l-arginine as an obligate intermediate. Although much progress has been made in recent years in the elucidation of the reaction mechanism of NOS, many questions remain to be answered. The use of low temperature has been instrumental in the revelation of the mechanism of NO synthesis, particularly regarding the role of the cofactor 5,6,7,8-tetrahydrobopterin (BH4). High-pressure studies may be expected to be similarly useful, but have been very few so far. In this short review, we depict the present state of knowledge about the reaction mechanism of NO synthesis, and the role(s) BH4 plays in it. This exposition is followed by a summary of the results obtained thus far in high-pressure studies and of the conclusions that can be drawn from them.

Animals↗

PubMed2005

Modulation of prion protein structure by pressure and temperature.

High pressure and temperature have been used efficiently to shed light on prion protein structure and folding. These physical parameters induce different conformational states of the prion protein, suggesting that prion structural changes occur within a complex energy landscape. Pressure has been used to prevent and even reverse prion protein aggregation. Alternatively, depending on experimental conditions, pressure also promotes prion protein aggregation leading to the formation of amorphous aggregates and amyloid fibrils. The latter ones show all characteristics of the pathogenic scrapie form. Furthermore, the pressure effects on prion protein structure appear to be strongly dependent on the integrity of the disulfide bond. In this paper, we discuss the mechanism and the origin of these opposing effects of pressure, taking the truncated form of hamster prion protein (SHaPrP(90-231)) as a model.

Disulfides↗

PubMed2005

Active sites of two orthologous cytochromes P450 2E1: differences revealed by spectroscopic methods.

Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics.

Animals↗

PubMed2005

Hunting oxygen complexes of nitric oxide synthase at low temperature and high pressure.

The reaction of nitric oxide synthase (NOS) with oxygen is fast and takes place within several steps, separated by ephemeral intermediates. The use of extreme experimental conditions, such as low temperature and high pressure, associated to rapid kinetic analysis, has proven to be a convenient tool to study this complex reaction. Stopped-flow experiments under high pressure indicated that oxygen binding occurred in more than one step. This was further corroborated by the detection of two short-lived oxy-compounds, differing in their spectral and electronic properties. Oxy-I resembles the ferrous oxygen complex known for cytochrome P450, whereas oxy-II appears to be locked in the superoxide form. Subzero temperature spectroscopy, together with an analytical separation method, revealed that the subsequent one-electron reduction of the oxygen complex is carried out by the NOS cofactor tetrahydrobiopterin (BH4). The low-temperature stabilized oxidation product of BH4 was found to be a protonated BH3 radical. Finally, work in the presence of a BH4 analog indicated that proton transfer to the activated oxygen complex is a second essential function of BH4.

Animals↗

PubMed2005

Tetrahydrobiopterin as combined electron/proton donor in nitric oxide biosynthesis: cryogenic UV-Vis and EPR detection of reaction intermediates.

The role of tetrahydrobiopterin (BH4) as a cofactor in nitric oxide synthase (NOS) has been the object of intense research in the last few years. It was found that in addition to its established effects on the NOS heme spin state, substrate affinity, and enzyme dimerization, BH4 is required as a one-electron donor to oxyferrous [Fe(II).O2] heme that is formed as an intermediate in the catalytic cycle. Cryogenic spectroscopic techniques proved particularly useful in the identification of this role of BH4 in NO synthesis. With these methods, the mechanism of fast reactions, such as the reaction of ferrous NOS with O2, can be unraveled by lowering the reaction temperature to subzero values. This may not only reduce the rate to such an extent that the reaction can be followed on a time scale from seconds to minutes, but intermediates may be observed that do not accumulate at higher temperatures. Cryogenic ultraviolet-visible (UV-vis) and electron paramagnetic resonance spectroscopy have been applied to clarify why the BH4 analogue 4-amino-tetrahydrobiopterin (4-amino-BH4) is unable to support NO synthesis. In the course of these studies, evidence was gathered supporting a role for BH4 as an obligate proton and electron donor. It is believed that the inhibitory action of 4-amino-BH4 derives from an inability to serve as a proton donor, even though it is perfectly able to serve as an electron donor. In this chapter, the suitability, drawbacks, and advantages of cryogenic methods are discussed.

Biopterins↗

PubMed2005

The role of the 132-160 region in prion protein conformational transitions.

The 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. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a beta-sheet form.

Amino Acids↗

PubMed2004

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II.

Pressure-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 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.

Computer Simulation↗

PubMed2004

High pressure induces scrapie-like prion protein misfolding and amyloid fibril formation.

Our 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. Within the "protein-only hypothesis", the reason for this hindrance may be that the experimental conditions used did not allow selection of the pathway adopted in vivo resulting in conversion into the infectious form. Here, using a pressure perturbation approach, we show that recombinant PrP is converted to a novel misfolded conformer, which is prone to aggregate and ultimately form amyloid fibrils. A short incubation at high pressure (600 MPa) of the truncated form of hamster prion protein (SHaPrP(90-231)) resulted in the formation of pre-amyloid structures. The mostly globular aggregates were characterized by ThT and ANS binding, and by a beta-sheet-rich secondary structure. After overnight incubation at 600 MPa, amyloid fibrils were formed. In contrast to pre-amyloid structures, they showed birefringency of polarized light after Congo red staining and a strongly decreased ANS binding capacity, but enhanced ThT binding. Both aggregate types were resistant to digestion by PK, and can be considered as potential scrapie-like forms or precursors. These results may be useful for the search for compounds preventing pathogenic PrP misfolding and aggregation.

Amyloid↗

PubMed2004

Histidine-tryptophan-ketoglutarate solution for organ preservation in human liver transplantation-a prospective multi-centre observation study.

Based on experimental work and clinical small studies, histidine-tryptophan-ketoglutarate (HTK) solution was found to be suitable not only for heart and kidney preservation but also for liver preservation. We decided, therefore, to use this preservation solution for clinical liver preservation in a prospective multi-centre trial. Enrolment to the study was from 1996 to 1999 in four European centres, and the results of 214 patients with HTK-preserved organs were analysed. Analysis showed a primary dysfunction (PDF) rate of 8.8%, with a primary non-function (PNF) rate of 2.3% and initial poor function (IPF) in 6.5%. Patient survival rate at 1 year was 83% and 1-year graft survival rate was 80%. In a univariate and a multivariate analysis PDF, early surgical complications and tendentiously severe infections (septicaemia, pneumonia, cholangitis) were identified as independent risk factors for graft and patient survival. Preservation with HTK can be regarded as an established alternative to preservation with University of Wisconsin (UW) solution when preservation times are short. Definitive assessment of the efficacy of preservation solutions requires further prospective randomised clinical trials that compare HTK and UW.

Drug Therapy, Combination↗

PubMed2004

Evidence of two distinct oxygen complexes of reduced endothelial nitric oxide synthase.

Oxygen binding to the oxygenase domain of reduced endothelial nitric oxide synthase (eNOS) results in two distinct species differing in their Soret and visible absorbance maxima and in their capacity to exchange oxygen by CO. At 7 degrees C, heme-oxy I (with maxima at 420 and 560 nm) is formed very rapidly (k(on) approximately 2.5.10(6) m(-1).s(-1)) in the absence of substrate but in the presence of pterin cofactor. It is capable of exchanging oxygen with CO at -30 degrees C. Heme-oxy II is formed more slowly (k(on) approximately equal to 3.10(5) m(-1).s(-1)) in the presence of substrate, regardless of the presence of pterin. It is also formed in the absence of both substrate and pterin. In contrast to heme-oxy I, it cannot exchange oxygen with CO at cryogenic temperature. In the presence of arginine, heme-oxy II is characterized by absorbance maxima near 432, 564, and 597 nm. When arginine is replaced by N-hydroxyarginine, and also in the absence of both substrate and pterin, its absorbance maxima are blue-shifted to 428, 560, and 593 nm. Heme-oxy I seems to resemble the ferrous dioxygen complex observed in many hemoproteins, including cytochrome P450. Heme-oxy II, which is the oxygen complex competent for product formation, appears to represent a distinct conformation in which the electronic configuration is essentially locked in the ferric superoxide complex.

Animals↗

PubMed2004

CO exchange of the oxyferrous complexes of endothelial nitric-oxide synthase oxygenase domain in the presence of 4-amino-tetrahydrobiopterin.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a 1-electron donor to the oxyferrous-heme complex. 4-Amino-tetrahydrobiopterin (4-amino-BH4) inhibits NO synthesis, although it has similar redox properties. We recently reported that 4-amino-BH4 is capable of electron transfer to Fe(II).O(2) in cryogenic single-turnover [J. Biol. Chem. 278 (2003) 48602]. We also suggested that BH4 serves as a proton donor to the Fe(II).O(2)(-) complex, and that 4-amino-BH4 cannot perform this second essential function. To corroborate these claims and to further characterize the intermediates observed after oxygenation of NOS in the presence of 4-amino-BH4, we added CO immediately after O(2) addition to the reduced oxygenase domain of endothelial NOS at -30 degrees C. This resulted in complete formation of a P450-type Fe(II).CO complex with either Arg or NG-hydroxy-L-arginine as the substrate. In the presence of 4-amino-BH2, which is redox-inactive, the same procedure yielded ferric heme with either substrate, without formation of any Fe(II).CO complex. We conclude: (i) O(2) binding to ferrous heme in the presence of 4-amino-BH2 is essentially irreversible; (ii) 4-amino-BH4 can reduce the oxyferrous complex; (iii) O(2)(-), rather than H(2)O(2) is the immediate product of uncoupled catalysis in the presence of 4-amino-BH4.

Animals↗

PubMed2004

Use of hydrostatic pressure to produce 'native' monomers of yeast enolase.

The 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. If the experiments are performed in the presence of 1 mm Mg(2+), or with apo-enzyme, the enzyme is inactivated [Kornblatt, M.J., Lange R., Balny C. (1998) Eur. J. Biochem 251, 775-780]. The UV spectra of the high pressure forms of the Mg(2+)- and apo-forms of enolase are identical and distinct from the spectrum of the form obtained in the presence of 1 mm Mn(2+); this suggests that Mn(2+) remains bound to the high pressure form of enolase. With Mn-enolase, the various spectral changes do not occur in the same pressure range, indicating that multiple processes are occurring. Pressure experiments were performed as a function of [Mn(2+)] and [protein]. One of the changes in the UV spectra shows a dependence on protein concentration, indicating that enolase is dissociating into monomers. The small changes in the UV spectrum and the retention of activity lead to a model in which enolase, in the presence of high concentrations of Mn(2+), dissociates into native monomers; upon release of pressure, the enzyme is fully active. Although further spectral changes occur at higher pressures, there is no inactivation as long as Mn(2+) remains bound. We propose that the relatively small and polar nature of the subunit interface of yeast enolase, including the presence of several salt bridges, is responsible for the ability of hydrostatic pressure to dissociate this enzyme into monomers with a native-like structure.

Enzyme Stability↗

PubMed2003

My passion for extreme conditions to solve the cytochrome P450 and NO synthase reaction mechanisms.

Cytochrome P450, and especially its reaction mechanism, has always been a major research interest of Professor I.C. Gunsalus. The reaction cycle of this enzyme is complex, containing many elementary steps and intermediates, and constitutes a challenge for the scientific community. During his repeated stays in our laboratories in Paris and in Montpellier, he contributed decisively to our study of the P450 reaction mechanism under extreme conditions, i.e., at subzero temperatures and at high pressure. From this initial impulse, we continued the work with different forms of cytochrome P450 and later on with nitric oxide synthase. This paper gives an overview of the insights into these enzymes gained by the use of extreme conditions. These exciting achievements were initiated by numerous discussions with Professor Gunsalus and also reflect a long-lasting collaboration and friendship.

Camphor 5-Monooxygenase↗