Thermodynamics of the alkaline transition in phytocyanins

The thermodynamics of the alkaline transition which influences the spectral and redox properties of the type 1 copper center in phytocyanins has been determined spectroscopically. The proteins investigated include Rhus vernicifera stellacyanin, cucumber basic protein and its Met89Gln variant, and umecyanin, the stellacyanin from horseradish roots, along with its Gln95Met variant. The changes in reaction enthalpy and entropy within the protein series show partial compensatory behavior. Thus, the reaction free energy change (hence the pK _a value) is rather variable. This indicates that species-dependent differences in reaction thermodynamics, although containing an important contribution from changes in the hydrogen-bonding network of water molecules in the hydration sphere of the protein (which feature enthalpy–entropy compensation), are to a large extent protein-based. The data for axial ligand variants are consistent with the hypothesis of a copper-binding His as the deprotonating residue responsible for this transition.     

Structural transition of bovine plasma albumin in the alkaline region--the N-B transition

Bovine plasma albumin (BPA) has approximately one SH group (Cys-34) which catalyzes the intramolecular SH, S-S exchange reaction in the alkaline region at low ionic strength, resulting in the formation of the aged form. So, the N-B transition at ionic strength above 0.20 and below 0.10 was studied using BPA and iodoacetamide-blocked BPA (IA-BPA), respectively. (1) pH profiles of [theta]262 and [theta]268 of BPA in 0.20 M KCl showed the characteristic changes in the pH region 7.0-9.0, corresponding to the N-B transition. On going from pH 7.0 to 9.0 in 0.10 M KCl or NaCl, IA-BPA did not show significant changes in rotational relaxation times of tryptophyl fluorophors, CD-resolved secondary structures, spin-echo 1H-n.m.r. spectra and cross-relaxation times (TIS) between irradiated and observed protein protons, which might reflect the rigidity of the domains and/or subdomains. On the other hand, rotational relaxation times of 1-anilino-8-naphthalenesulfonate-IA-BPA complex (IA-BPA-ANS0.9, molar ratio of ANS to IA-BPA = 0.9/1) showed significant decreases from 131 to 114 ns on going from the N- to the B-forms in 0.10 M KCl. The above results and reported experimental evidence might indicate that on going from the N- to the B-forms in 0.10 M KCl or NaCl, the mutual movement of subdomains, connected with a flexible hinge region (Brown & Shockley (1982)) might increase without loss in the helicity and the rigidity of subdomains. (2) The N-B transition of IA-BPA in the absence of salt was quite different from those in 0.10 M KCl or NaCl. Decreases in the helicity and the intramolecular rigidity, as monitored by TIS-measurements, were observed on going from the N- to the B-forms.     

Mutation-induced perturbation of the cytochrome c alkaline transition

The possible influence of residue Phe-82 in the cytochrome c alkaline isomerization has been evaluated by spectrophotometric pH titrations of a family of mutant yeast iso-1-cytochromes c in which the identity of the residue at this position has been varied. The pKa for the exchange of the Met-80 heme iron ligand was determined from pH titrations in which the S----Fe charge-transfer band (695 nm) was monitored and was found to be 8.5 for the wild type, 7.7 for Ser-82, 7.7 for Gly-82, 7.2 for Leu-82, and 7.2 for Ile-82. pH-jump experiments [Davis et al. (1974) J. Biol. Chem. 249, 2624] established that substitutions at position 82 affect the alkaline isomerization by lowering the pKa of the titrating group by as much as 1.4 pK units; for the Ser-82 and Gly-82 variants, there is also a small effect on the Keq for the ligand exchange equilibrium. On the basis of these findings, we conclude that one critical role for Phe-82 in the wild-type protein is stabilization of the native heme binding environment.     

The alkaline transition of swine pepsinogen

At alkaline pH, swine pepsinogen is reversibly inactivated in a transition which involves the cooperative release of two protons from the molecule and is governed by a pK = 9. Stopped flow kinetic studies on the absorbance changes accompanying this reaction show that it can be resolved into two steps, with increasing pH; a slow conformational change, whose amplitude follows the ionisation curve of one group of pK = 9.9, followed by a rapid pH dependent conformational change, linked to a group of pK = 8.2. The pH dependence of the rate of the slow step is interpreted to show the presence of a protonated group which cannot ionise in the neutral form of the zymogen, but is in slow equilibrium with a form where it titrates with a pK = 6.8. At the same time, a histidine in the amino terminal region of the protein becomes reactive to diethyl pyrocarbonate, suggesting this to be the group which triggers the reaction.     

Thermodynamics of the alkaline transition of cytochrome c.

The apparent equilibrium constant (Kapp) of the alkaline transition (AT) of beef heart cytochrome c, obtained from pH titrations of the current intensities in cyclic voltammetry experiments, has been measured as a function of the temperature from 5 to 65 degrees C, at different ionic strength (I = 0.01-0.2 M). The temperature profile of the pKapp values is biphasic and yields two distinct sets of DeltaH degrees 'AT and DeltaS degrees 'AT values below and above approximately 40 degrees C. In the low-temperature range, the process is endothermic and is accompanied by a small positive entropy change, while at higher temperatures it becomes less endothermic and involves a pronounced entropy loss. The temperature dependence of the transition thermodynamics is most likely the result of the thermal transition of native ferricytochrome c from a low-T to an high-T conformer which occurs at alkaline pH values at a temperature comparable with above (Ikeshoji, T., Taniguchi, I., and Hawkridge, F. M. (1989) J. Electroanal. Chem. 270, 297-308; Battistuzzi, G., Borsari, M., Sola, M., and Francia, F. (1997) Biochemistry 36, 16247-16258). Thus, it is apparent that the transitions of the two native conformers to the corresponding alkaline form(s) are thermodynamically distinct processes. It is suggested that this difference arises from either peculiar transition-induced changes in the hydration sphere of the protein or to the preferential binding of different lysines to the heme iron in the two temperature ranges. Extrapolation of the Kapp values at null ionic strength allowed the determination of the thermodynamic equilibrium constants (Ka) at each temperature, hence of the "true" standard thermodynamic parameters of the transition. The pKa value at 25 degrees C was found to be 8.0. A pKapp value of 14.4 was calculated for the alkaline transition of ferrocytochrome c at 25 degrees C and I = 0.1 M. The much greater relative stabilization of the native state in the reduced as compared to the oxidized form turns out to be almost entirely enthalpic in origin, and is most likely due to the greater affinity of the methionine sulfur for the Fe(II) ion. Finally, it is found that the Debye-Hückel theory fits the ionic strength dependence of the pKapp values, at least qualitatively, as observed previously for the ionic strength dependence of the reduction potential of this protein class. It is apparent that the increase in the pKapp values with increasing ionic strength is for the most part an entropic effect.     

Folding units govern the cytochrome c alkaline transition

The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.     

DNA helix-coil transition in alkaline medium

Dna helix-coil transition in th alkaline medium was considered theoretically and experimentally. On the basis of the theory and experimental comparison the DNA double-stranded form deprotonation was revealed.     

The alkaline transition of swine pepsinogen.

At alkaline pH, swine pepsinogen is reversibly inactivated in a transition which involves the cooperative release of two protons from the molecule and is governed by a pK = 9. Stopped flow kinetic studies on the absorbance changes accompanying this reaction show that it can be resolved into two steps, with increasing pH; a slow conformational change, whose amplitude follows the ionisation curve of one group of pK = 9.9, followed by a rapid pH dependent conformational change, linked to a group of pK = 8.2. The pH dependence of the rate of the slow step is interpreted to show the presence of a protonated group which cannot ionise in the neutral form of the zymogen, but is in slow equilibrium with a form where it titrates with a pK 6.8. At the same time, a histidine in the amino terminal region of the protein becomes reactive to diethyl pyrocarbonate, suggesting this to be the group which triggers the reaction.     

The alkaline transition of turnip peroxidases.

A stopped-flow kinetic study of the spectral changes which appear during the transition between the neutral and the alkaline form of two turnip peroxidases P₁ and P₇ was conducted. With P₁, the kinetics of the spectral changes present three distinct steps: One is fast while the other two are slow. From the pH dependence of the observed rate constant for the fast step, it is proposed that this step might represent deprotonation by the hydroxide anions of a heme-linked group with a pK of 10.1. For P₇, only one step was detected which was fast. In this case, the pH dependence of the observed rate constant indicates that a heme-linked group is deprotonated either by water solvent molecules or by hydroxide anions. The simplest explanation for the observed results is that the groups titrated represent a water molecule in the sixth coordination position of the iron for both peroxidases. The small values of the rate constants found for these deprotonation reactions are explained in terms of hydroxide anion binding to the sixth coordination position of the iron or by the existence of a negatively charged electrostatic gate which prevents negatively charged ligands from entering the heme pocket. A reanalysis of the results reported for a similar study with horseradish peroxidase shows that the alkaline transition of this hemoprotein can also be explained in the same manner as for turnip peroxidases.     

A model of the transition state in the alkaline phosphatase reaction

A high resolution crystal structure of Escherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 A resolution. The vanadate ion takes on a trigonal bipyramidal geometry and is covalently bound by the active site serine nucleophile. A coordinated water molecule occupies the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the vanadate ion are stabilized by interactions with both Arg-166 and the zinc metal ions of the active site. This structural complex supports the in-line displacement mechanism of phosphomonoester hydrolysis by alkaline phosphatase and provides a model for the proposed transition state in the enzyme-catalyzed reaction.     

Optical spectroscopic investigation of the alkaline transition in umecyanin from horseradish root

Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His(2)Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N(epsilon2)H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.     

Investigating cat predation as the cause of bat wing tears using forensic DNA analysis

Cat predation upon bat species has been reported to have significant effects on bat populations in both rural and urban areas. The majority of research in this area has focussed on observational data from bat rehabilitators documenting injuries, and cat owners, when domestic cats present prey. However, this has the potential to underestimate the number of bats killed or injured by cats. Here, we use forensic DNA analysis techniques to analyze swabs taken from injured bats in the United Kingdom, mainly including Pipistrellus pipistrellus (40 out of 72 specimens). Using quantitative PCR, cat DNA was found in two‐thirds of samples submitted by bat rehabilitators. Of these samples, short tandem repeat analysis produced partial DNA profiles for approximately one‐third of samples, which could be used to link predation events to individual cats. The use of genetic analysis can complement observational data and potentially provide additional information to give a more accurate estimation of cat predation.     

Mitochondrial DNA mutations cause resistance to opening of the permeability transition pore

The age-related accumulation of mitochondrial DNA mutations has the potential to impair organ function and contribute to disease. In support of this hypothesis, accelerated mitochondrial mutagenesis is pathogenic in the mouse heart, and there is an increase in myocyte apoptosis. The current study sought to identify functional alterations in cell death signaling via mitochondria. Of particular interest is the mitochondrial permeability transition pore, opening of which can initiate cell death, while pore inhibition is protective. Here, we show that mitochondria from transgenic mice that develop mitochondrial DNA mutations have a marked inhibition of calcium-induced pore opening. Temporally, inhibited pore opening coincides with disease. Pore inhibition also correlates with an increase in Bcl-2 protein integrated into the mitochondrial membrane. We hypothesized that pore inhibition was mediated by mitochondrial Bcl-2. To test this hypothesis, we treated isolated mitochondria with Bcl-2 antagonistic peptides (derived from the BH3 domain of Bax or Bid). These peptides released the inhibition to pore opening. The data are consistent with a Bcl-2-mediated inhibition of pore opening. Thus, mitochondrial DNA mutations induce an adaptive-protective response in the heart that inhibits opening of the mitochondrial permeability pore.     

Identification of the ligand-exchange process in the alkaline transition of horse heart cytochrome c.

Magnetic-circular-dichroism (m.c.d.) spectra over the wavelength range 300-2000 nm at room temperature and at 4.2K of horse heart cytochrome c are reported at a series of pH values between 7.8 and 11.0, encompassing the alkaline transition. The effect of glassing agents on the e.p.r. spectrum at various pH values is also reported. Comparison of these results with spectra obtained for the n-butylamine adduct of soybean leghaemoglobin support the hypothesis that lysine is the sixth ligand in the alkaline form of horse heart cytochrome c. The m.c.d. and e.p.r. spectra of horse heart cytochrome c in the presence of 1-methylimidazole have also been examined. These studies strongly suggest that histidine-18, the proximal ligand of the haem, is the ionizing group that triggers the alkaline transition. Low-temperature m.c.d. and e.p.r. spectra are also reported for Pseudomonas aeruginosa cytochrome c551. It is shown that no ligand exchange takes place at the haem in this species over the pH range 6.0-11.3.     

Dimethylaminopyridine derivatives of lupane triterpenoids cause mitochondrial disruption and induce the permeability transition

Triterpenoids are a large class of naturally occurring compounds, and some potentially interesting as anticancer agents have been found to target mitochondria. The objective of the present work was to investigate the mechanisms of mitochondrial toxicity induced by novel dimethylaminopyridine (DMAP) derivatives of pentacyclic triterpenes, which were previously shown to inhibit the growth of melanoma cells in vitro. MCF-7, Hs 578T and BJ cell lines, as well as isolated hepatic mitochondria, were used to investigate direct mitochondrial effects. On isolated mitochondrial hepatic fractions, respiratory parameters, mitochondrial transmembrane electric potential, induction of the mitochondrial permeability transition (MPT) pore and ion transport-dependent osmotic swelling were measured. Our results indicate that the DMAP triterpenoid derivatives lead to fragmentation and depolarization of the mitochondrial network in situ, and to inhibition of uncoupled respiration, induction of the permeability transition pore and depolarization of isolated hepatic mitochondria. The results show that mitochondrial toxicity is an important component of the biological interaction of DMAP derivatives, which can explain the effects observed in cancer cells.     

Investigating the link between epithelial-mesenchymal transition and the cancer stem cell phenotype: A mathematical approach

Under the cancer stem cell (CSC) hypothesis, sustained metastatic growth requires the dissemination of a CSC from the primary tumour followed by its re-establishment in a secondary site. The epithelial-mesenchymal transition (EMT), a differentiation process crucial to normal development, has been implicated in conferring metastatic ability on carcinomas. Balancing these two concepts has led researchers to investigate a possible link between EMT and the CSC phenotype—indeed, recent evidence indicates that, following induction of EMT in human breast cancer and related cell lines, stem cell activity increased, as judged by the presence of cells displaying the CD44^high/CD24^low phenotype and an increase in the ability of cells to form mammospheres. We mathematically investigate the nature of this increase in stem cell activity. A stochastic model is used when small number of cells are under consideration, namely in simulating the mammosphere assay, while a related continuous model is used to probe the dynamics of larger cell populations. Two scenarios of EMT-mediated CSC enrichment are considered. In the first, differentiated cells re-acquire a CSC phenotype—this model implicates fully mature cells as key subjects of de-differentiation and entails a delay period of several days before de-differentiation occurs. In the second, pre-existing CSCs experience accelerated division and increased proportion of self-renewing divisions; a lack of perfect CSC biomarkers and cell sorting techniques requires that this model be considered, further emphasizing the need for better characterization of the mammary (cancer) stem cell hierarchy. Additionally, we suggest the utility of comparing mammosphere data to computational mammosphere simulations in elucidating the growth characteristics of mammary (cancer) stem cells.     

Free energy of transition for the individual alkaline conformers of yeast iso-1-cytochrome c

Direct protein electrochemistry was used to obtain the thermodynamic parameters of transition from the native (state III) to the alkaline (state IV) conformer for untrimethylated Saccharomyces cerevisiae iso-1-cytochrome c expressed in E. coli and its single and multiple lysine-depleted variants. In these variants, one or more of the lysine residues involved in axial Met substitution (Lys72, Lys73, and Lys79) was mutated to alanine. The aim of this work is to determine the thermodynamic affinity of each of the substituting lysines for the heme iron and evaluate the interplay of enthalpic and entropic factors. The equilibrium constants for the deprotonation reaction of Lys72, 73, and 79 were computed for the minimized MD average structures of the wild-type and mutated proteins, applying a modified Tanford-Kirkwood calculation. Solvent accessibility calculations for the substituting lysines in all variants were also performed. The transition enthalpy and entropy values within the protein series show a compensatory behavior, typical of a process involving extensive solvent reorganization effects. The experimental and theoretical data indicate that Lys72 most readily deprotonates and replaces M80 as the axial heme iron ligand, whereas Lys73 and Lys79 show comparably higher pKa values and larger transition free energies. A good correlation is found within the series between the lowest calculated Lys pKa value and the corresponding experimental pKa value, which can be interpreted as indicative of the deprotonating lysine itself acting as the triggering group for the conformational transition. The triple Lys to Ala mutant, in which no lysine residues are available for heme iron binding, features transition thermodynamics consistent with a hydroxide ion replacing the axial methionine ligand.     

Functional role of a protein foldon--an Omega-loop foldon controls the alkaline transition in ferricytochrome c

Hydrogen exchange results for cytochrome c and several other proteins show that they are composed of a number of foldon units which continually unfold and refold and account for some functional properties. Previous work showed that one Omega-loop foldon controls the rate of the structural switching and ligand exchange behavior of cytochrome c known as the alkaline transition. The present work tests the role of foldons in the alkaline transition equilibrium. We measured the effects of denaturant and 14 destabilizing mutations. The results show that the ligand exchange equilibrium is controlled by the stability of the same foldon unit implicated before. In addition, the results obtained confirm the epsilon-amino group of Lys79 and Lys73 as the alkaline replacement ligands and bear on the search for a triggering group.     

Quenching of the intrinsic fluorescence of liver alcohol dehydrogenase by the alkaline transition and by coenzyme binding

The fluorescence of alcohol dehydrogenase is quenched by the acid dissociation of some group on the protein having an apparent pKa of 9.6 at 25 degrees C. The pKa of this alkaline quenching transition is unchanged by the binding of trifluoroethanol or pyrazole to the enzyme or by the selective removal of the active site of Zn2+ ion. This indicates that the ionization of a zinc-bound water molecule is not responsible for the quenching. The binding of NAD+ to the enzyme causes a drop in protein fluorescence and an apparent shift in the alkaline quenching transition to lower pH. In the ternary complex formed with NAD+ and trifluoroethanol the alkaline transition is difficult to discern between pH 6 and pH 11. In the NAD+-pyrazole ternary complex, however, a small but noticeable fluorescence transition is observed with a pKa(app) approximately 9.5. We propose that the alkaline transition centered at pH 9.6 is not shifted to lower pH upon binding NAD+. Instead, the amplitude of the alkaline quenching effect is decreased to the point that it is difficult to detect when NAD+ is bound. We present a model that describes the dependence of the fluorescence of the protein on pH and NAD+ concentration in terms of two independently operating, dynamic quenching mechanisms. Our data and model cast serious doubt on the identification, made previously in the literature, between the alkaline quenching pKa and the pKa of the group whose ionization is coupled to NAD+ binding.(ABSTRACT TRUNCATED AT 250 WORDS)