Nuclear magnetic resonance studies of the unfolding of pancreatic ribonuclease. II. Unfolding by urea and guanidine hydrochloride.

The unfolding of ribonuclease by urea and guanidine hydrochloride has been studied by ¹H nuclear magnetic resonance spectroscopy, under conditions where the unfolding is fully reversible and concentration-independent. Both urea and guanidine produce marked changes in the chemical shift of the histidine C₍₂₎H resonances, together with small changes in other regions of the spectrum, at concentrations (0.1 to 1.0 m) far below those which are required for gross unfolding of the protein. The changes in area of the histidine C₍₂₎H resonances through the major unfolding transition produced by these denaturants give evidence for the existence of at least two intermediates in the unfolding process. The “order of unfolding” of the histidine residues is closely similar for both urea and guanidine hydrochloride unfolding, and also similar to that found for thermal unfolding at low pH (see Benz &; Roberts, 1975) accompanying paper.     

The aromatic residues of bovine pancreatic ribonuclease studied by 1H nuclear magnetic resonance.

1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.     

Solvent Microenvironments and Copper Binding Alters the Conformation and Toxicity of a Prion Fragment

The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu²⁺ ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu²⁺ supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu²⁺ ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu²⁺ ions, owing to binding affinity of copper towards histidine moiety present in the peptide.     

External Ba2+ block of human Kv1.5 at neutral and acidic pH: evidence for Ho+-induced constriction of the outer pore mouth at rest

Previous studies have shown that low pH_o accelerates depolarization-induced inactivation and decreases the macroscopic conductance by reducing channel availability. To test the hypothesis that outer pore constriction underlies the decreased conductance at low pH_o, external Ba²⁺ was used to examine the accessibility of the channel pore at rest under neutral and acidic conditions. At pH_o 7.4, Ba²⁺ block of closed channels follows a monoexponential time course and involves a low-affinity superficial site (K_D ≅ 1 mM, −80 mV, 0 mM ) and a high-affinity site (K_D ≅ 4 μM) deeper in the pore. Depolarization promotes Ba²⁺ dissociation and an analytical model incorporating state-dependent changes of Ba²⁺ affinity is presented that replicates the frequency dependence of the time course and the extent of block. Open-channel block by Ba²⁺ is weak. At pH_o 5.5, both the access to and exit from the deep site is inhibited. These results are consistent with a low-pH_o-induced conformational change in the outer pore that prevents Ba²⁺ binding at rest or unbinding during depolarization. If a pore constriction is involved, similar to that proposed to occur during P/C-type inactivation, this would imply that closed-state inactivation in Kv1.5 occurs under acidic conditions.     

Serpin polymerization is prevented by a hydrogen bond network that is centered on his-334 and stabilized by glycerol

Polymerization of serpins commonly results from mutations in the shutter region underlying the bifurcation of strands 3 and 5 of the A-sheet, with entry beyond this point being barred by a H-bond network centered on His-334. Exposure of this histidine in antithrombin, which has a partially opened sheet, allows polymerization and peptide insertion to occur at pH 6 or less when His-334 will be predictably protonated with disruption of the H-bond network. Similarly, thermal stability of antithrombin is pH-dependent with a single unfolding transition at pH 6, but there is no such transition when His-334 is buried by a fully closed A-sheet in heparin-complexed antithrombin or in alpha(1)-antitrypsin. Replacement of His-334 in alpha(1)-antitrypsin by a serine or alanine at pH 7.4 results in the same polymerization and loop-peptide acceptance observed with antithrombin at low pH. The critical role of His-334 and the re-formation of its H-bond network by the conserved P8 threonine, on the full insertion of strand 4, are relevant for the design of therapeutic blocking agents. This is highlighted here by the crystallographic demonstration that glycerol, which at high concentrations blocks polymerization, can replace the P8 threonine and re-form the disrupted H-bond network with His-334.     

Hydrogen exchange of the tryptophan residues in bovine alpha-lactalbumin studied by UV spectroscopy

The effect of Ca²⁺ ion on structural fluctuation of a milk Ca²⁺-binding protein, α-lactalbumin, under native conditions was investigated by comparing hydrogen-exchange reactions of tryptophan residues in the apo-form without Ca²⁺ and in the holo-form at 1 mM CaCl₂ at pH 7.0 in the presence of 0.1M Na⁺. The reactions were followed by measuring time-dependent absorption changes at 298–300 nm due to the ²H-¹H exchange of the tryptophan imino protons and were found to be biphasic under all the conditions examined. Two of the four tryptophan protons are insensitive to Ca²⁺ concentration and show a relatively fast exchange rate. The other two protons are much more extensively protected (a protection degree of 10³–10⁵) and are markedly affected by the presence of Ca²⁺. Examinations of the temperature dependence and pH dependence of the individual exchange rates have been utilized for elucidating the exchange mechanism. The fast protons show a low activation energy reaction with so-called EX₂ kinetics. The exchange reaction of the slow protons is accompanied by a high activation energy, and the exchange mechanism of the protons depended on the presence or absence of stabilizing Ca²⁺ ions—the EX₁ kinetics for the apo-protein and the EX₂ kinetics for the holo-protein at 1 mM Ca²⁺. The exchange reaction in the thermally unfolded state was also found to be biphasic, but the fast phase, which has an exchange rate in the fully exposed state, becomes predominant with decreasing temperature. By taking this fact and using a structural unfolding model of hydrogen exchange, the present results are fully consistent with thermodynamic parameters of the thermal transition and kinetic parameters of refolding reactions induced by concentration jumps of guanidine hydrochloride obtained in previous studies. It is demonstrated that the reaction of the slow protons in the native state is mediated by a transient global unfolding equivalent to the “thermal” unfolding under a native condition and that switching of the exchange mechanism from the EX₁ to EX₂ kinetics results from acceleration of the refolding rate with an increase in Ca²⁺ concentration. The transient global unfolding takes place even under a strongly native condition, e.g., at a temperature 20° below the beginning of the thermal transition.     

Comparative studies of the interaction of human and bovine platelet factor 4 with heparin using histidine NMR resonances as spectroscopic probes

ThepK _a values of His-38 and His-50 of the heparin-binding protein, bovine platelet factor 4, are 5.6 and 6.5, respectively, as determined by¹H NMR spectroscopy. The¹H NMR resonance of His-38 of bovine platelet factor 4 which exhibits the lowerpK _a value is perturbed upon heparin binding to a greater degree than the resonance of His-50. Human platelet factor 4 contains the homologous residues His-23 and His-35. ThepK _a values of the two histidine residues of human platelet factor 4 are 5.3 and 6.4. The¹H NMR resonance of the histidine of human platelet factor 4 exhibiting the lowerpK _a value also is perturbed upon heparin binding to a greater degree than the histidine resonance exhibiting the higherpK _a , thereby suggesting comparable heparin-protein interactions in bovine and human platelet factor 4.     

Comparison of the helix–coil conformational changes of poly(deoxyadenylate-deoxytymidylate) and poly(deoxyadenylate-deoxythymidylate) induced by variations of hydrogen-ion concentration

Double-helical poly(dG-dC) and poly(dA-dT) are DNA analogs in which the interactions between the two strands of the helix are, respectively, either the stronger G/C type or the weaker A/T type along the entire length of macromolecules. Thus, these synthetic polynucleotides can be considered as representatives of the most stable and the least stable DNA. In the investigations presented here, potentiometric titrations and stopped-flow kinetic experiments were carried out in order to compare the pH-induced helix–coil conformations (10°C and 150mM [Na⁺]) the pH of the helix–coil transition (pH_m) is 12.81 for poly(dG-dC) and 11.76 for poly(dA-dT). The unwinding of double-helical poly(dG-dC) initiated by a sudden change in pH was found to be a simple exponential process with rate constants in the range of 200–600 sec^?1, depending on the final value of the pH jump. The intramolecular double-helix formation of poly(dG-dC) was studied by lowering the pH of the solutions from a value above pH_m to that below pH_m in dilute solutions (15.5 ug/ml [polymer]). Under these conditions, the observed rewinding reactions displayed a major and two exponential phases, all of which were independent of polymer concentration. From the comparison of the results of poly(dA-dT) and poly(dG-dT) would unwind faster than poly(dG-dC). However, if the pH jumps are such that they present the same perturbation of these polymers relative to their pH_m values, no significant differences exist between the rates of helix–coil conformation changes of poly(dA-dT) and poly(dG-dC).     

p NMR Studies of Rat Liver Cold Preservation with Histidine-Buffered Lactobionate Solution

The efficiency of a preservation medium, histidine-buffered lactobionate solution (HBLS), was determined by measuring post-ischemic recoveries of ATP and intracellular pH under Krebs-Henseleit buffer (KHB) perfusion. We used NMR spectroscopy to study the effect of 24-h cold ischemia, followed by 4°C then 37°C reperfusion on the isolated rat liver. Three media were compared: University of Wisconsin solution (UW-lactobionate); Bretschneider's solution (HTK); HBLS and HBLS supplemented with 2 mM Gly and 2 mM Cys (HBLSg2) or with 10 mM Gly and 2 mM Cys (HBLSg10). All values were compared to control values measured during pre-ischemic cold perfusion with KHB (ATP = 8.60 ± 0.6 μmol/g of dry weigh and pH_in = 7.41 ± 0.05). The main result from ³¹p NMR data concerned ATP recovery during cold reperfusion, which was significantly higher in the HBLS group (112 ± 10%) as compared to the UW and HTK groups (around 66%). The presence of glycine decreased ATP recovery (88 ± 8% in HBLSg2, 79 ± 15% in HBLSg10). Higher values of recovered pH_in were observed in livers stored in histidine buffered solutions (around 7.30) as compared to UW (around 7.20); histidine was by ¹³C NMR proved to accumulate in the liver cells, thus ensuring a good buffering capacity. The thermal transition induced a decrease in both ATP level and pH_in in all groups. This might be the result of a stimulation of the carbohydrate metabolism (as demonstrated by ¹³C NMR) especially when glycine was present in the storage solution.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H⁺ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H⁺-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H⁺ in the course of cellular amino acid uptake by PAT1.     

Thermodynamics of transfer ribonucleic acids: the effect of sodium on the thermal unfolding of yeast tRNAPhe.

The thermal unfolding of yeast phenylalanine-specific tRNA (tRNA^Phe) has been calorimetrically investigated at several salt concentrations in the absence of magnesium. Application of the deconvolution theory of macromolecular conformational transitions allows calculation of the thermodynamic parameters of unfolding. It is demonstrated that the unfolding of tRNA^Phe occurs in a sequential fashion and that four separate transitions or five macromolecular thermodynamic states exist in the temperature range 8–72°C under the experimental conditions of these studies (0.067–0.52M Na⁺). The enthalpy and entropy changes between states and the relative population of each state as a function of temperature and salt concentration have been obtained. Sodium stabilizes the low-temperature conformations of tRNA^Phe. The increase in the melting temperatures of each transition is shown to be linearly dependent on the logarithm of sodium concentration. These results allow calculation of the “phase” diagram for the transitions as a function of salt concentration.     

The regulatory factor SipA is a highly stable β-II class protein with a SH3 fold

The small regulator SipA, interacts with the ATP-binding domain of non-bleaching sensor histidine kinase (NblS), the most conserved histidine kinase in cyanobacteria. NblS regulates photosynthesis and acclimation to a variety of environmental conditions. We show here that SipA is a highly stable protein in a wide pH range, with a thermal denaturation midpoint of 345 K. Circular dichroism and 1D ¹H NMR spectroscopies, as well as modelling, suggest that SipA is a β-II class protein, with short strands followed by turns and long random-coil polypeptide patches, matching the SH3 fold. The experimentally determined m-value and the heat capacity change upon thermal unfolding (ΔC_p) closely agreed with the corresponding theoretical values predicted from the structural model, further supporting its accuracy.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (T_m ≈ 45 °C) than does the kidney enzyme (T_m ≈ 55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Random coil chemical shift for intrinsically disordered proteins: effects of temperature and pH

Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins. The quality of the secondary chemical shifts is dependent on an appropriate choice of random coil chemical shifts. We report random coil chemical shifts and sequence correction factors determined for a GGXGG peptide series following the approach of Schwarzinger et al. (J Am Chem Soc 123(13):2970–2978, 2001). The chemical shifts are determined at neutral pH in order to match the conditions of most studies of intrinsically disordered proteins. Temperature has a non-negligible effect on the ¹³C random coil chemical shifts, so temperature coefficients are reported for the random coil chemical shifts to allow extrapolation to other temperatures. The pH dependence of the histidine random coil chemical shifts is investigated in a titration series, which allows the accurate random coil chemical shifts to be obtained at any pH. By correcting the random coil chemical shifts for the effects of temperature and pH, systematic biases of the secondary chemical shifts are minimized, which will improve the reliability of detection of transient secondary structure in disordered proteins.     

1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects

Summary In this study we report on the ¹H, ¹³C and ¹⁵N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of ¹H, ¹³C and ¹⁵N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strougly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.     

Nuclear-magnetic-resonance study of the histidine residues of S-peptide and S-protein and kinetics of 1H-2H exchange of ribonuclease A.

1H NMR spectroscopy at 100 MHz was used to determine the first-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35 degrees C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35 degrees C and pH meter reading 11 caused irreversible denaturation of RN-ase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem, (Tokyo) 75, 1197-1200 (1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed. The first-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5 The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936-937]. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26 degrees C. His-48 in S-protein titrates normally and has a lower pK than in RN-ase-A probably because of the absence of Asp-14, which in RN-ase-A forms a a hydrogen bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9.     

Heme coordination states of unfolded ferrous cytochrome C

The structural changes of ferrous Cyt-c that are induced by binding to SDS micelles, phospholipid vesicles, DeTAB, and GuHCl as well as by high temperatures and changes in the pH have been studied by RR and UV-Vis absorption spectroscopies. Four species have been identified in which the native methionine-80 ligand is removed from the heme iron. This coordination site is either occupied by a histidine (His-33 or His-26) to form a 6cLS configuration, which is the prevailing species in GuHCl at pH 7.0 and ambient temperature, or remains vacant to yield a 5cHS configuration. The three identified 5cHS species differ with respect to the hydrogen-bond interactions of the proximal histidine ligand (His-18) and include a nonhydrogen-bonded, a hydrogen-bonded, and a deprotonated imidazole ring. These structural motifs have been found irrespective of the unfolding conditions used. An unambiguous spectroscopic distinction of these 5cHS species is possible on the basis of the Fe-N(imidazole) stretching vibrations, the RR bands in the region between 1300 and 1650 cm(-1), and the electronic transitions in the Soret- and Q-band regions. In acid and neutral solutions, the species with a hydrogen-bonded and a nonhydrogen-bonded His-18 prevail, whereas in alkaline solutions a configuration with a deprotonated His-18 ligand is also observed. Upon lowering the pH or increasing the temperature in GuHCl solutions, the structure on the proximal side of the heme is perturbed, resulting in a loss of the hydrogen-bond interactions of the His-18 ligand. Conversely, the hydrogen-bonded His-18 of ferrous Cyt-c is stabilized by electrostatic interactions which increase in strength from phospholipid vesicles to SDS micelles. The results here suggest that unfolding of Cyt-c is initiated by the rupture of the Fe-Met-80 bond and structural reorganizations on the distal side of the heme pocket, whereas the proximal part is only affected in a later stage of the denaturation process.     

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

Summary The intracellular pH (pH _i ) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H⁺ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH _i of cells placed in an acidic medium (pH _o below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH _o is acutely reduced to 5.5, pH _i falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H⁺. The unidirectional influx of H⁺ exhibits saturation kinetics with respect to extracellular [H⁺]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK _m is 0.74 ± 0.09 × 10^–6 m.Steady-state cells with pH _i above 6.8 continuously extrude H⁺ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH _i 6.3) when returned to pH _o 7.3 medium respond by transporting H⁺, resulting in a rapid rise in pH _i . The halftime for this process is 1.09 ± 0.22 min. The H⁺ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH _i to its steady-state value.     

Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H⁺ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H⁺ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH_o) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH_i) and net H⁺ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH_i remained above 7.0 within a pH_o range of 4 to 6 and decreased below pH_o 4. Above pH_o 4, H⁺ extrusion increased correspondingly, with the maximum value at pH_o 5.5, and below pH_o 4, a net H⁺ influx was observed. Without glucose in the medium, the pH_i decreased, and a net H⁺ influx was observed below pH_o 5.5. A high correlation (R = 0.75 to 0.92) between the pH_i and net H⁺ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH_i homeostasis in L. monocytogenes subjected to acid stress.     

Urea induced unfolding of F isomer of human serum albumin: a case study using multiple probes

The human serum albumin is known to undergo N <==> F (neutral to fast moving) isomerization between pH 7 and 3.5. The N < ==> F isomerization involves unfolding and separation of domain III from rest of the molecule. The urea denaturation of N isomer of HSA shows two step three state transition with accumulation of an intermediate state around 4.8-5.2 M urea concentration. While urea induced unfolding transition of F isomer of HSA does not show the intermediate state observed during unfolding of N isomer. Therefore, it provides direct evidence that the formation of intermediate in the unfolding transition of HSA involves unfolding of domain III. Although urea induced unfolding of F isomer of HSA appears to be an one step process, but no coincidence between the equilibrium transitions monitored by tryptophanyl fluorescence, tyrosyl fluorescence, far-UV CD and near-UV CD spectroscopic techniques provides decisive evidence that unfolding of F isomer of HSA is not a two state process. An intermediate state that retained significant amount of secondary structure but no tertiary structure has been identified (around 4.4 M urea) in the unfolding pathway of F isomer. The emission of Trp-214 (located in domain II) and its mode of quenching by acrylamide and binding of chloroform indicate that unfolding of F isomer start from domain II (from 0.4 M urea). But at higher urea concentration (above 1.6 M) both the domain unfold simultaneously and the protein acquire random coil structure around 8.0 M urea. Further much higher KSV of NATA (17.2) than completely denatured F isomer (5.45) of HSA (8.0 M urea) suggests the existence of residual tertiary contacts within local regions in random coil conformation (probably around lone Trp-214).