Biomedical subjects

C Balny

Publications and source records attributed to C Balny.

At least 19 recordsLinked to original sources

PubMed2006

Pressure-jump-induced kinetics reveals a hydration dependent folding/unfolding mechanism of ribonuclease A.

Pressure-jump (p-jump)-induced relaxation kinetics was used to explore the energy landscape of protein folding/unfolding of Y115W, a fluorescent variant of ribonuclease A. Pressure-jumps of 40 MPa amplitude (5 ms dead-time) were conducted both to higher (unfolding) and to lower (folding) pressure, in the range from 100 to 500 MPa, between 30 and 50 degrees C. Significant deviations from the expected symmetrical protein relaxation kinetics were observed. Whereas downward p-jumps resulted always in single exponential kinetics, the kinetics induced by upward p-jumps were biphasic in the low pressure range and monophasic at higher pressures. The relative amplitude of the slow phase decreased as a function of both pressure and temperature. At 50 degrees C, only the fast phase remained. These results can be interpreted within the framework of a two-dimensional energy surface containing a pressure- and temperature-dependent barrier between two unfolded states differing in the isomeric state of the Asn-113-Pro-114 bond. Analysis of the activation volume of the fast kinetic phase revealed a temperature-dependent shift of the unfolding transition state to a larger volume. The observed compensation of this effect by glycerol offers an explanation for its protein stabilizing effect.

Animals↗

PubMed2005

The powerful high pressure tool for protein conformational studies.

The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2 degrees, 3 degrees and 4 degrees structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.

Ataxin-3↗

PubMed2004

High pressure, an alternative approach to understand protein misfolding diseases.

Protein folding is essential for the flow of genetic information to biological activity. A failure in this process can result in disease, by causing cell damage and sometimes death. The misfolding of proteins often induces their aggregation, initiating the fibril formation seen in a range of human and animal diseases. Because misfolding and aggregation are of fundamental importance in vivo, there is currently great interest in understanding their mechanisms. To gain insight into the folding and unfolding processes of proteins, for nearly a century, an original biophysical approach has been successfully used: the application of high hydrostatic pressure combined with various spectroscopic and kinetic techniques. Because high pressure provides new insight into protein structure and folding which cannot be obtained by other techniques, the conformations of pressure-induced unfolding intermediates and species involved in the initial states of aggregation of proteins associated with specific diseases are currently being investigated. Our contention is that by exploring folding kinetics, misfolding pathways and stability under pressure, it will be possible to understand the mechanisms of amyloidogenesis, with the ultimate goal to design therapeutic strategies to prevent progression of the disease.

Amyloid↗

PubMed2004

Characterization of a complex formed between human plasminogen and recombinant sheep prion: pressure and thermal sensitivity of complex formation.

Scrapie is thought to be caused by one or more conformations of a proteinacious particle called a prion. The infectious form(s) is referred to as the scrapie form of the prion protein (PrPsc) whereas a benign form, the cellular conformer, is referred to as PrPC. The cellular conformation of the sheep prion protein formed a 1:1 complex with human plasminogen. The complex precipitated at 0 degrees C (Ksp = 17* 10(-12) M2). This precipitation reaction was sensitive to both temperature and pressure. When subjected to hydrostatic pressure the precipitate dissolved. At 25 degrees C the complex was soluble with a dissociation constant of about 10(-7) M as determined by isothermal titration calorimetry. Absorption, fluorescence and circular dichroism spectroscopy showed that neither protein, in the complex, underwent a detectable structural change so long as proteolytic inhibitors were present. In the absence of proteolytic inhibitors, plasminogen slowly cleaved the prion protein.

Animals↗

PubMed2001

Protein folding pathways of adenylate kinase from E. coli: hydrostatic pressure and stopped-flow studies.

Adenylate kinase (AKe) from E. coli is a small, single-chain, monomeric enzyme with no tryptophan and a single cysteine residue. We have constructed six single-Trp mutants of AKe to facilitate optical studies of these proteins and to specifically examine the interrelationship between their structure, function, dynamics, and folding reactions. In this study, the effects of hydrostatic pressure on the folding reactions of AKe were studied. The native structure of AKe was transformed to a non-native, yet pressure stable, conformation by hydrostatic pressure of about 300 MPa. This pressure lability of AKe is rather low for a monomeric protein and presumably may be attributed to substantial conformational flexibility and a correspondingly large volume change. The refolding of AKe after pressure-induced denaturation was reversible under ambient conditions. At low temperature (near 0 degrees C), the refolding process of pressure-exposed AKe mutants displayed a significant hysteresis. The observation of a slow refolding rate in the 193 region and a faster folding rate around the active site (86, 41, 73 regions) leads us to suggest that in the folding process, priority is afforded to functional regions. The slow structural return of the 193 region apparently does not hinder the more rapid return of enzymatic activity of AKe. Circular dichroism studies on the pressure-denatured Y193W mutant show that the secondary structure (calculated from far-UV spectra) returned at a rapid rate, but the tertiary structure alignment (calculated from near-UV spectra) around the 193 region occurred more slowly at rates comparable to those detected by fluorescence intensity. Denaturation of AKe mutants by guanidine hydrochloride and subsequent refolding experiments were also consistent with a much slower refolding process around the 193 region than near the active site. Fast refolding kinetic traces were observed in F86W, S41W, and A73W mutants using a fluorescence detection stopped-flow rapid mixing device, while only a slow kinetic trace was observed for Y193W. The results suggest that the differences in regional folding rates of AKe are not derived from the specific denaturation methods, but rather are inherent in the structural organization of the protein.

Adenylate Kinase↗

PubMed2001

High pressure refolding of recombinant human growth hormone from insoluble aggregates. Structural transformations, kinetic barriers, and energetics.

Two different types of insoluble, non-native aggregates of recombinant human growth hormone were formed by agitation in buffer or buffer containing 0.75 m guanidine HCl (GdnHCl) and characterized by infrared and second derivative UV spectroscopies. The degree of secondary structural perturbation was greater in the aggregates formed in 0.75 m GdnHCl. Both aggregate types were dissolved and refolded using high hydrostatic pressures in combination with either elevated temperature or non-denaturing levels of guanidine HCl or urea. The effects of a range of temperature, pressure, and chaotrope concentrations were tested and led to optimal conditions that approached 100% yield of native protein. The aggregates formed in 0.75 m GdnHCl required higher concentrations of urea or GdnHCl, or higher temperature or pressure for a yield equivalent to that for aggregates formed in buffer alone. Investigation of the effects of pressure, temperature, and chaotrope on unfolding of rhGH documented that under conditions used for optimal high pressure disaggregation and refolding, the native state is greatly favored thermodynamically (e.g. 25 kJ/mol at 2000 bar and 0.75 m GdnHCl). Dissolution of aggregates under pressure is a kinetically limited process. Comparison of refolding yields in GdnHCl and urea solutions suggest that pressure effects on electrostatic interactions do not dominate pressure effects on disaggregation. We suggest that non-native hydrogen bonds between protein molecules within aggregates of recombinant human growth hormone are responsible for the rate-limiting kinetic barrier in pressure-induced disaggregation.

Dose-Response Relationship, Drug↗

PubMed2001

Pressure denaturation of the yeast prion protein Ure2.

Denaturation of the Saccharomyces cerevisiae prion protein Ure2 was investigated using hydrostatic pressure. Pressures of up to 600 MPa caused only limited perturbation of the structure of the 40-kDa dimeric protein. However, nondenaturing concentrations of GdmCl in combination with high pressure resulted in complete unfolding of Ure2 as judged by intrinsic fluorescence. The free energy of unfolding measured by pressure denaturation or by GdmCl denaturation is the same, indicating that pressure does not induce dimer dissociation or population of intermediates in 2 M GdmCl. Pressure-induced changes in 5 M GdmCl suggest residual structure in the denatured state. Cold denaturation under pressure at 200 MPa showed that unfolding begins below -5 degrees C and Ure2 is more susceptible to cold denaturation at low ionic strength. Results obtained using two related protein constructs, which lack all or part of the N-terminal prion domain, were very similar.

Cold Temperature↗

PubMed2001

Pressure denaturation of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

The effects of hydrostatic pressure on apo wild-type glyceraldehyde-3-phosphate dehydrogenase (wtGAPDH) from Bacillus stearothermophilus (B. stearothermophilus) have been studied by fluorescence spectroscopy under pressure from 0.1 to 650 MPa. Unlike yeast GAPDH [Ruan, K. C., and Weber, G. (1989) Biochemistry 28, 2144-2153], denaturation of the tetrameric apo wtGAPDH from B. stearothermophilus is likely to precede dissociation into subunits. As expected, denaturation is accompanied by the loss of enzymatic activity. B. stearothermophilus apo wtGAPDH interfaces are less pressure sensitive than apo yeast GAPDH ones, while NAD does not protect B. stearothermophilus wtGAPDH against denaturation by pressure. The pressure effects on B. stearothermophilus GAPDH whose R and Q-axis interfaces were destabilized by disruption of interfacial hydrogen bonds are similar to that of apo wtGAPDH.

Apoenzymes↗

PubMed2001

Pressure-exploration of the 33-kDa protein from the spinach photosystem II particle.

The 33-kDa protein isolated from the spinach photosystem II particle is an ideal model to explore high-pressure protein-unfolding. The protein has a very low free energy as previously reported by chemical unfolding studies, suggesting that it must be easy to modulate its unfolding transition by rather mild pressure. Moreover, the protein molecule consists of only one tryptophan residue (Trp241) and eight tyrosine residues, which can be conveniently used to probe the protein conformation and structural changes under pressure using either fluorescence spectroscopy or fourth derivative UV absorbance spectroscopy. The different experimental methods used in the present study indicate that at 20 degrees C and pH 6, the 33-kDa protein shows a reversible two-state unfolding transition from atmospheric pressure to about 180 MPa. This value is much lower than those found for the unfolding of most proteins studied so far. The unfolding transition induces a large red shift of the maximum fluorescence emission of 34 nm (from 316 nm to 350 nm). The change in standard free energy (DeltaGo) and in volume (DeltaV) for the transition at pH 6.0 and 20 degrees C are -14.6 kJ.mol-1 and -120 mL.mol-1, respectively, in which the DeltaGo value is consistent with that obtained by chemical denaturation. We found that pressure-induced protein unfolding is promoted by elevated temperatures, which seem largely attributed to the decrease in the absolute value of DeltaGo (only a minor variation was observed for the DeltaV value). However, the promotion of the unfolding by alkaline pH seems mainly related to the increase in DeltaV without any significant changes in DeltaGo. It was also found that NaCl significantly protects the protein from pressure-induced unfolding. In the presence of 1 M NaCl, the pressure needed to induce the half-unfold of the protein is shifted to a higher value (shift of 75 MPa) in comparison with that observed without NaCl. Interestingly, in the presence of NaCl, the value of DeltaV is significantly reduced whilst that of DeltaGo remains as before. The unfolding-refolding kinetics of the protein has also been studied by pressure-jump, in which it was revealed that both reactions are a two-state transition process with a relatively slow relaxation time of about 102 s.

Anilino Naphthalenesulfonates↗

PubMed2001

Reversible changes of the wheat gamma 46 gliadin conformation submitted to high pressures and temperatures.

The structure of the wheat gamma 46 gliadin was investigated, in aqueous solutions, under high pressure or temperature by the use of ultraviolet and fluorescence spectroscopic techniques. We found that high pressure (above 400 MPa) induces a change in the protein conformation that results in a decrease of the polarity of the environment of aromatic amino acids. This new conformation was able to bind the hydrophobic probe, 8-anilino-1-naphtalene-sulfonic acid (ANS), indicating an increase in the gliadin surface hydrophobicity. Thermodynamic parameters of this conformational change were measured and infrared spectroscopy studies were used to probe the potential secondary structure modifications. The high stability of gamma 46 gliadin could be related to its elastic character, as the observed changes were always found to be reversible.

Anilino Naphthalenesulfonates↗

PubMed2000

Pressure effects on tryptophan and its derivatives.

The high pressure effects on fluorescence of free tryptophan (Trp) and its derivatives, N-acetyl-tryptophan (AT), N-acetyl-tryptophanamide (NATA), tryptophanamide (TA), and tryptophan, containing 6-polypeptides in aqueous solution, were investigated in a pressure range from 0.1 to 650 MPa. It was found by analyzing the center of spectral mass in the wavelength range from 300 to 450 nm that high pressure shifted the fluorescence spectra of all these species to red direction: 421 cm(-1) for Trp, 305 cm(-1) for AT, 310 cm(-1) for NATA, 265 cm(-1) for TA, and 220 cm(-1) for single tryptophan containing 6-polypeptides. All the fluorescence efficiencies (i.e., quantum yield) of the compounds were reduced with pressure except free tryptophan where its fluorescence efficiency was enhanced with pressure. Glycerol, ethanol, and pH obviously influenced the pressure effects on their fluorescence characteristics. Since the tryptophan fluorescence is usually used as a probe for protein structural investigation, these findings suggested that the intrinsic pressure effect on tryptophan (or its derivatives) must be taken in consideration to explain the phenomenon observed in high pressure study on biomolecules when using the usual fluorospectroscopic approaches. In the present investigation, the mechanisms involved for pressure effects on tryptophan and its derivatives were explored and discussed.

Ethanol↗

PubMed2000

Inactivation of creatine kinase by high pressure may precede dimer dissociation.

The effects of hydrostatic pressure on creatine kinase activity and conformation were investigated using either the high-pressure stopped-flow method in the pressure range 0.1-200 MPa for the activity determination, or the conventional activity measurement and fluorescence spectroscopy up to 650 MPa. The changes in creatine kinase activity and intrinsic fluorescence show a total or partial reversibility after releasing pressure, depending on both the initial value of the high pressure applied and on the presence or absence of guanidine hydrochloride. The study on 8-anilinonaphthalene-1-sulfonate binding to creatine kinase under high pressure indicates that the hydrophobic core of creatine kinase was progressively exposed to the solvent at pressures above 300 MPa. This data shows that creatine kinase is inactivated at low pressure, preceding both the enzyme dissociation and the unfolding of the hydrophobic core occurring at higher pressure. Moreover, in agreement with the recently published structure of the dimer, it can be postulated that the multistate transitions of creatine kinase induced both by pressure and guanidine denaturation are in direct relationship with the existence of hydrogen bonds which maintain the dimeric structure of the enzyme.

Anilino Naphthalenesulfonates↗

PubMed2000

Effects of hydrostatic pressure on the structure and biological activity of infectious bursal disease virus.

The effects of high hydrostatic pressure on the structure and biological activity of infectious bursal disease virus (IBDV), a commercially important pathogen of chickens, were investigated. IBDV was completely dissociated into subunits at a pressure of 240 MPa and 0 degrees C revealed by the change in intrinsic fluorescence spectrum and light scattering. The dissociation of IBDV showed abnormal concentration dependence as observed for some other viruses. Electron microscopy study showed that morphology of IBDV had an obvious change after pressure treatment at 0 degrees C. It was found that elevating pressure destroyed the infectivity of IBDV, and a completely pressure-inactivated IBDV could be obtained under proper conditions. The pressure-inactivated IBDV retained the original immunogenic properties and could elicit high titers of virus neutralizing antibodies. These results indicate that hydrostatic pressure provides a potential physical means to prepare antiviral vaccine.

Anilino Naphthalenesulfonates↗

PubMed1999

Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin.

The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0. 1-700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 degrees C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are -78 mL.mol-1 and +73 mL.mol-1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ.mol-1 for the denaturation and -5 kJ. mol-1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.

Anilino Naphthalenesulfonates↗

PubMed1999

The effects of hydrostatic pressure on the conformation of plasminogen.

Plasminogen undergoes a large conformational change when it binds 6-aminohexanoate. Using ultraviolet absorption spectroscopy and native PAGE, we show that hydrostatic pressure brings about the same conformational change. The volume change for this conformational change is -33 mL.mol-1. Binding of ligand and hydrostatic pressure both cause the protein to open up to expose surfaces that had previously been buried in the interior.

Aminocaproic Acid↗

PubMed1999

Hydrostatic pressure and calcium-induced dissociation of calpains.

The dissociation of mu- and m-calpains was studied by fluorescence spectroscopy under high hydrostatic pressure (up to 650 MPa). Increasing pressure induced a red shift of the tryptophan fluorescence of the calcium-free enzyme. The concentration dependence of the spectral transition was consistent with a pressure-induced dissociation of the subunits. Rising temperature increased the stability of calpain heterodimers and confirmed the predominance of hydrophobic interactions between monomers. At saturating calcium, the spectral transition was not observed for native or iodoacetamide-inactivated calpains, indicating that they were already dissociated by calcium. The reaction volume was about -150 ml mol-1 for both isoforms, and the dissociation constants at atmospheric pressure are approximately 10-12 M and 10-15 M for mu- and m-calpains, respectively. This result indicates a tighter interaction in the isoform that requires higher calcium concentration for activity.

Calcium↗

PubMed1999

Pressure regulation of malic dehydrogenase in reversed micelles.

Malic dehydrogenase (MDH) studied in water and reversed micelles upon pressure application revealed a difference in catalysis. Whereas MDH in water appeared to be not sensitive to the pressure increasing, the catalytic activity of MDH in reversed micelles showed bell-shaped dependencies both on pressure and surfactant hydration degree, w0. The catalytic activity of MDH was found to be maximal under moderate pressure equal to 300-500 bar and at w0 approximately 14 with the difference between lowest and highest levels of the catalytic activity amounted to about 10 times. The work presented demonstrates for the first time the co-operative effect of reversed micelles and pressure application to malic dehydrogenase leading to the enzyme regulation that cannot be realized in aqueous solution.

Catalysis↗

PubMed1999

Contribution of the carbohydrate moiety to conformational stability of the carboxypeptidase Y high pressure study.

The process of pressure-induced denaturation of carboxypeptidase Y and the role of the carbohydrate moiety in its response to pressure and low temperature were investigated by measuring in situ the catalytic activity and, the intrinsic and 8-anilino-1-naphthalene sulfonic acid binding fluorescences. Pressure-induced denaturation of carboxypeptidase Y is a process involving at least three transitions. Low pressures (below 150 MPa) induced slight conformational changes characterized by a slight decrease in the center of the spectral mass of intrinsic fluorescence, whereas no changes in 8-anilino-1-naphthalene sulfonic acid binding fluorescence were observed and 80% of the catalytic activity remained. Higher pressure (150-500 MPa) induced further conformational changes, characterized by a large decrease in the center of the spectral mass of intrinsic fluorescence, a large increase in the 8-anilino-1-naphthalene sulfonic acid binding fluorescence and the loss of all catalytic activity. Thus, this intermediate exhibited characteristics of molten globule-like state. A further increase, in pressure (above 550 MPa) induced transition from this first molten globule-like state to a second molten globule-like state. This two-stage denaturation process can be explained by assuming the existence of two independent structural domains in the carboxypeptidase molecule. A similar three-transition process was found for unglycosylated carboxypeptidase Y, but, the first two transitions clearly occurred at lower pressures than those for glycosylated carboxypeptidase Y. These findings indicate that the carbohydrate moiety protects carboxypeptidase Y against pressure-induced denaturation. The origin of the protective effects is discussed based on the known crystallographic structure of CPY.

Carboxypeptidases↗