Acidic pH-induced conformational changes in amyloidogenic mutant transthyretin

Several proteins, including transthyretin (TTR), can generate in tissues extracellular insoluble aggregates, in the form of fibrils, that are associated with pathological states known as amyloidoses. To date, more than 80 different TTR point mutations have been associated with hereditary amyloidosis in humans. In vitro, the formation of amyloid fibrils by human TTR is known to be triggered by acidic pH. We show here that, in vitro, the natural amyloidogenic I84S and the non-natural I84A TTR mutant forms exhibit a propensity to produce fibrils in an acidic medium significantly higher than that of wild-type TTR. The two mutant forms have been crystallized at both neutral and acidic pH. Their neutral pH crystal structures are very similar to that of wild-type TTR, consistent with previous evidence indicating that only minor structural changes are induced by amyloidogenic mutations. On the contrary, their crystal structures at moderately low pH (4.6) show significant conformational differences as compared to their neutral pH structures. Remarkably, such changes are not induced in wild-type TTR crystallized at low pH. The most relevant consist of the unwinding of the TTR short alpha-helix and of the change in conformation of the loop connecting the alpha-helix to beta-strand F. Only one monomer of the crystallographic dimer is affected, causing a disruption of the tetrameric symmetry. This asymmetry and a possible destabilization of the tetrameric quaternary structure of TTR may be responsible for the amyloidogenic potential of the two TTR mutant forms at low pH.     

Aminosulfonate modulated pH-induced conformational changes in connexin26 hemichannels

Gap junction channels regulate cell-cell communication by passing metabolites, ions, and signaling molecules. Gap junction channel closure in cells by acidification is well documented; however, it is unknown whether acidification affects connexins or modulating proteins or compounds that in turn act on connexins. Protonated aminosulfonates directly inhibit connexin channel activity in an isoform-specific manner as shown in previously published studies. High-resolution atomic force microscopy of force-dissected connexin26 gap junctions revealed that in HEPES buffer, the pore was closed at pH < 6.5 and opened reversibly by increasing the pH to 7.6. This pH effect was not observed in non-aminosulfonate buffers. Increasing the protonated HEPES concentration did not close the pore, indicating that a saturation of the binding sites occurs at 10 mM HEPES. Analysis of the extracellular surface topographs reveals that the pore diameter increases gradually with pH. The outer connexon diameter remains unchanged, and there is a approximately 6.5 degrees rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca2+-induced closure.     

Acid pH-induced conformational changes in bovine liver rhodanese.

The enzyme rhodanese is greatly stabilized in the range pH 4-6, and samples at pH 5 are fully active after several days at 23 degrees C. This is very different from results at pH greater than 7, where there is significant loss of activity within 1 h. A pH-dependent conformational change occurs below pH 4 in a transition centered around pH 3.25 that leads slowly to inactive rhodanese at pH 3 (t 1/2 = 22 min at pH3). The inactive rhodanese can be reactivated by incubation under conditions required for detergent-assisted refolding of denatured rhodanese. The inactive enzyme at pH 3 has the maximum of its intrinsic fluorescence spectrum shifted to 345 nm from 335 nm, which is characteristic of native rhodanese at pH greater than 4. At pH 3, rhodanese shows increased exposure of organized hydrophobic surfaces as measured by 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid binding. The secondary structure is maintained over the entire pH range studied (pH 2-7). Fluorescence anisotropy measurements of the intrinsic fluorescence provide evidence suggesting that the pH transition produces a state that does not display greatly increased average flexibility at tryptophan residues. Pepsin digestibility of rhodanese follows the pH dependence of conformational changes reported by activity and physical methods. Rhodanese is resistant to proteolysis above pH 4 but becomes increasingly susceptible as the pH is lowered. The form of the enzyme at pH 3 is cleaved at discrete sites to produce a few large fragments. It appears that pepsin initially cleaves close to one end of the protein and then clips at additional sites to produce species of a size expected for the individual domains into which rhodanese is folded. Overall, it appears that in the pH range between pH 3 and 4, titration of groups on rhodanese leads to opening of the structure to produce a conformation resembling, but more rigid than, the molten globule state that is observed as an intermediate during reversible unfolding of rhodanese.     

Indications of pH-induced conformational changes in phosphofructokinase from baker's yeast.

The partition of phosphorfructokinase (EC 2.7.1.11) from baker's yeast between the liquid phases of an aqueous biphasic systeem changes drastically in the pH interval 7-8, in contrast to other proteins. This abnormal behaviour is correlated to changes in sedimentation coefficient and binding capacity of the enzyme in this pH region. Since the molecular weight of phosphofructokinase does not change, these findings must reflect conformational changes in the enzyme molecule.     

pH-induced conformational changes in the soluble manganese-stabilizing protein of photosystem II

In this paper, we analyzed the pH-induced changes in the conformational states of the manganese-stabilizing protein (MSP) of photosystem II. Distinct conformational states of MSP were identified using fluorescence spectra, far-UV circular dichroism, and pressure-induced unfolding at varying suspension pH values, and four different conformational states of MSP were clearly distinguished using the center of fluorescence spectra mass when suspension pH was altered from 2 to 12. MSP was completely unfolded at a suspension pH above 11 and partly unfolded below a pH of 3. Analysis of the center of fluorescence spectral mass showed that the MSP structure appears stably folded around pH 6 and 4. The conformational state of MSP at pH 4 seems more stable than that at pH 6. Studies of peak positions of tryptophan fluorescence and MSP-bound 1-anilinonaphthalene-8-sulfonic acid fluorescence spectra supported this observation. A decrease in the suspension pH to 2 resulted in significant alterations in the MSP structure possibly because of protonation of unprotonated residues at lower pH, suggesting the existence of a large number of unprotonated amino acid residues at neutral pH possibly useful for proton transport in oxygen evolution. The acidic pH-induced conformational changes of MSP were reversible upon increase of pH to neutral pH; however, N-bromosuccinimide modification of tryptophan (Trp241) blocks the recovery of pH-induced conformational changes in MSP, implying that Trp241 is a key residue for the unfolded protein to form a functional structure. Thus, pH-induced structural changes of stable MSP (pH 6-4) may be utilized to analyze its functionality as a cofactor for oxygen evolution.     

pH-induced conformational states of bovine growth hormone

The folding behavior of bovine growth hormone (bGH) is examined by chemical and pH denaturation using several spectroscopic probes of protein secondary and tertiary structure. Partially denaturing concentrations of urea eliminate the native-state quenching of intrinsic tryptophan fluorescence, from the single protein tryptophan, but the fluorescence emission spectrum is not red-shifted like the unfolded state, and the protein retains substantial secondary structure. A neutral-to-acid pH shift also eliminates tryptophan quenching; however, the loss of quenching is not accompanied by an emission red-shift. In addition, the protein undergoes a pH-dependent UV absorbance transition; the changes in absorptivity have the same midpoint as the transition associated with the change in intrinsic tryptophan fluorescence. The magnitude of the absorption transition is similar to that observed previously for urea denaturation of the protein. In a similar fashion, a pH-dependent CD transition is also observed; however, the transition occurs at a higher pH. The behavior of the various optical probes indicates that the pH-induced conformational transition produces a highly populated species in which the microenvironment surrounding the single protein tryptophan residue resembles that observed during the urea-induced unfolding/refolding transition. The pH-induced changes in tertiary structure occur at a lower pH than the changes associated with a portion of the secondary structure. Proton NMR of the low-pH intermediate indicates that the three His and six Tyr resonances are indistinguishable from the unfolded state. The intermediate(s) observed by either chemical or pH-induced denaturation resemble(s) a molten globule state which contains significant secondary structure. The residual secondary structure present in the intermediate could be nonnative.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-induced conformational changes in spinach ferredoxin: steady-state and time-resolved fluorescence studies.

Steady-state and time-resolved fluorescence techniques were used to monitor pH-induced conformational changes in spinach ferredoxin. An increase was seen in the wave-length maximum of tryptophan-73 (Trp-73) emission, from 325 nm below pH 6.0 to 342 nm above pH 7.0, indicating significantly diminished hydrophobicity, at pH 7.0, in the environment of the indole ring. Raising the solution pH from 6.0 to 7.6 also decreased the binding of the detergent Brij-96, showing that the ferredoxin molecule as a whole became more hydrophilic at higher pH. Nonionic (acrylamide) and ionic (I- and Cs+) quenchers were used to probe the tryptophan environment. Trp-73 is partially shielded from I-, presumably by negatively charged residues, as predicted from the amino acid sequence and three-dimensional structure of plant-type ferredoxins. Ionic strength and pH effects on tryptophan fluorescence lifetimes follow a pattern common to single-tryptophan proteins: the emission decays can be fit to a biexponential model in which the lifetime of the excited state increases with increasing pH. The indication of a pH-induced conformational change in the range pH 6.0 to 7.6 is discussed with reference to the physiological association of ferredoxin with ferredoxin:NADP+ oxidoreductase and the rise in chloroplast stromal pH in the light.     

Kinetics of the low pH-induced conformational changes and fusogenic activity of influenza hemagglutinin.

The decrease of the intrinsic tryptophan fluorescence intensity of purified influenza (X31 strain) hemagglutinin (HA) was used to monitor the low pH-induced conformational change of this protein. The kinetics of the fluorescence decrease depended strongly on the pH. At pH optimal for fusion, the change in tryptophan fluorescence was fast and could be fitted to a monoexponential function. We measured a rate constant of 5.78 s-1 (t1/2 = 120 ms) at pH 4.9 using rapid stopped-flow mixing. Under suboptimal conditions (higher pH), the rate constant was decreased by an order of magnitude. In addition, a slow component appeared and the fluorescence decrease followed a sum of two exponentials. The kinetics of conformational changes were compared with those of the fusion of influenza virus with red blood cell membranes as assessed by the R18-dequenching assay. At optimal pH the HA conformational change was not rate-limiting for the fusion process. However, at sub-optimal pH, the slow transition to the fusogenic conformational of HA resulted in slower kinetics and decreased extent of fusion.     

Low pH-induced conformational changes in vesicular stomatitis virus glycoprotein involve dramatic structure reorganization

Membrane fusion is the key step in the entry of enveloped animal viruses into their host cells. Fusion of vesicular stomatitis virus with membranes occurs at acidic pH and is mediated by its envelope glycoprotein, the G protein. To study the structural transitions induced by acidic pH on G protein, we have extracted the protein from purified virus by incubation with nonionic detergent. At pH 6.0, purified G protein was able to mediate fusion of either phospholipid vesicles or Vero cells in culture. Intrinsic fluorescence studies revealed that changes in the environment of Trp residues occurred as pH decreases. In the absence of lipidic membranes, acidification led to G protein aggregation, whereas protein-protein interactions were substituted by protein-lipid interactions in the presence of liposomes. 1,1'-Bis(4-aniline-5-naphthalene sulfonate) (bis-ANS) binding was utilized to probe the degree of exposure of hydrophobic regions of G protein during acidification. Bis-ANS binding was maximal at pH 6.2, suggesting that a hydrophobic segment is exposed to the medium at this pH. At pH 6.0, a dramatic decrease in bis-ANS binding was observed, probably due to loss of tridimensional structure during the conformational rearrangement. This hypothesis was confirmed by circular dichroism analysis at different pH values, which showed a great decrease in alpha-helix content at pH values close to 6.0, suggesting that a reorganization of G protein secondary structure occurs during the fusion reaction. Our results indicate that G protein undergoes dramatic structural changes at acidic pH and acquires a conformational state able to interact with the target membrane.     

pH-induced conformational changes in chromatin subunits measured by circular dichroism and immunochemical reactivity

Changes in the conformational state of chromatin core particles from chicken erythrocytes were studied by both immunochemical and biophysical methods as a function of pH and ionic strength. When the pH of core particles in a solution of ionic strength 3, 60 or 220 mM was lowered from pH 7.5, a sharp transition in the circular dichroism spectrum of DNA monitored between 320 and 260 nm was observed at pH 6.65. This change in DNA ellipticity was totally reversible. Binding to core particles of antibodies specific for histones H2B, H2A, H3 and for the IRGERA (synthetic C-terminal) peptide of H3 was used to follow changes in histone antigenicity. Binding was studied in the pH range 7.5-5.35, and at ionic strength of 60 and 220 mM. A change in reactivity of some histone epitopes was observed around pH 6.2–6.5. However, the changes observed by circular dichroism and antibody binding pertain to different components of chromatin subunits and they probably reflect independent phenomena. The alteration in accessibility of these determinants at the surface of core particles was completely reversible and was dependent on ionic strength. The conformation changes in core particles occurring near physiological ionic strength and pH may reflect dynamic changes in chromatin structure that possess functional significance.     

pH-induced conformational changes of AcrA, the membrane fusion protein of Escherichia coli multidrug efflux system

The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.     

Resolving the individual components of a pH-induced conformational change

This communication introduces a simple method to determine the pKs of microscopic ionizations from complex titration curves. We used this approach to study the alkaline transition (pH-dependent ligand exchange) of mitochondrial cytochrome c. The linearization of titration curves permitted resolution of two to three limiting microscopic ionizations. By combining these data with studies of the temperature dependence of ligand-exchange equilibria, we found evidence that the alkaline transition comprises two chemically distinct processes: the deprotonation of the alternative ligands and the break of the iron-methionine ligation bond. We also noted that, in the horse and untrimethylated S. cerevisiae iso-1 cytochromes c, the permissible deprotonation of the epsilon-amino group of Lys(72) allows formation of an alkaline isomer at lower pH, with lesser stability, which leads to hysteresis in the titration curves. The linearization of the titration curves for different cytochromes c thus brings insight on the microscopic contributions to conformational stability.     

pH-induced domain interaction and conformational transitions of lipoxygenase-1

The multidomain structure of soybean LOX1 was examined over the pH range 1-12. Lipoxygenase-1 activity was reversible over broad pH range of 4-10 due to the reversibility of conformational states of the molecule. Below pH 4.0, due to collapse in hydrophobic interactions, the enzyme unfolded to an irreversible conformation with the properties of molten globule state with a mid point of transition at pH 2.4. This intermediate state lost iron irreversibly. In alkaline pH at 11.5, LOX1 underwent partial unfolding with the exposure of cysteine residues with subsequent oxidation of a pair of cysteine residues in the C-terminal domain and this intermediate showed some properties of molten globule state and retained 35% of activity. Beyond pH 12.0, the enzyme was completely inactivated irreversibly due to irreversible conformational changes. The pH-dependent urea-induced unfolding of LOX1 suggested that LOX1 was more stable at pH 7.0 and least stable at pH 9.0. Furthermore, the urea-induced unfolding of LOX1 indicated that the unfolding was biphasic due to pH-dependent domain interactions and involved sequential unfolding of domains. The loss of enzyme activity at pH 4. 0 and 7.0 occurred much earlier to unfolding of the C-domain at all pHs studied. The combination of urea-induced unfolding measurements and limited proteolysis experiments suggested that at pH 4.0, the domains in LOX1 were less interactive and existed as tightly folded units. Furthermore, these results confirmed the contribution of ionic interactions in the interdomain contacts.     

Structural characterization and pH-induced conformational transition of full-length KcsA

The bacterial K+ channel KcsA from Streptomyces lividans was analyzed by neutron and x-ray small-angle solution scattering. The C-terminally truncated version of KcsA, amenable to crystallographic studies, was compared with the full-length channel. Analyzing the scattering data in terms of radius of gyration reveals differences between both KcsA species of up to 13.2 A. Equally, the real-space distance distribution identifies a 40 to 50 A extension of full-length KcsA compared to its C-terminally truncated counterpart. We show that the x-ray and neutron scattering data are amenable for molecular shape reconstruction of full-length KcsA. The molecular envelopes calculated display an hourglass-shaped structure within the C-terminal intracellular domain. The C-terminus extends the membrane spanning region of KcsA by 54-70 A, with a central constriction 10-30 A wide. Solution scattering techniques were further employed to characterize the KcsA channel under acidic conditions favoring its open conformation. The full-length KcsA at pH 5.0 shows the characteristics of a dumbbell-shaped macromolecular structure, originating from dimerization of the tetrameric K+ channel. Since C-terminally truncated KcsA measured under the same low pH conditions remains tetrameric, oligomerization of full-length KcsA seems to proceed via structurally changed C-terminal domains. The determined maximum dimensions of the newly formed complex increase by 50-60%. Shape reconstruction of the pseudooctameric complex indicates the pH-induced conformational reorganization of the intracellular C-terminal domain.     

Comparison of the pH-induced conformational change of different clostridial neurotoxins

Clostridial neurotoxins are internalized inside acidic compartments, wherefrom the catalytic chain translocates across the membrane into the cytosol in a low pH-driven process, reaching its proteolytic substrates. The pH range in which the structural rearrangement of clostridial neurotoxins takes place was determined by 8-anilinonaphthalene-1-sulfonate and tryptophan fluorescence measurements. Half conformational change was attained at pH 4.55, 4.50, 4.40, 4.60, 4.40, and 4.40 for tetanus neurotoxin and botulinum neurotoxin serotypes /A, /B, /C, /E, and /F, respectively. This similarity indicates the key residues for the conformation transition are strongly conserved. Acidic liposomes support the conformational rearrangement shifting the effect versus higher pH values, whereas zwitterionic liposomes do not. The disulfide bridge linking the light and the heavy chains together needs to be oxidized to allow toxin membrane insertion, indicating that in vivo its reduction follows exposure to the cytosol after penetration of the endosomal membrane.     

A model-structure of a periplasm-facing state of the NhaA antiporter suggests the molecular underpinnings of pH-induced conformational changes

The Escherichia coli NhaA antiporter couples the transport of H(+) and Na(+) (or Li(+)) ions to maintain the proper pH range and Na(+) concentration in cells. A crystal structure of NhaA, solved at pH 4, comprises 12 transmembrane helices (TMs), arranged in two domains, with a large cytoplasm-facing funnel and a smaller periplasm-facing funnel. NhaA undergoes conformational changes, e.g. after pH elevation to alkaline ranges, and we used two computational approaches to explore them. On the basis of pseudo-symmetric features of the crystal structure, we predicted the structural architecture of an alternate, periplasm-facing state. In contrast to the crystal structure, the model presents a closed cytoplasmic funnel, and a periplasmic funnel of greater volume. To examine the transporter functional direction of motion, we conducted elastic network analysis of the crystal structure and detected two main normal modes of motion. Notably, both analyses predicted similar trends of conformational changes, consisting of an overall rotational motion of the two domains around a putative symmetry axis at the funnel centers, perpendicular to the membrane plane. This motion, along with conformational changes within specific helices, resulted in closure at the cytoplasmic end and opening at the periplasmic end. Cross-linking experiments, performed between segments on opposite sides of the cytoplasmic funnel, revealed pH-dependent interactions consistent with the proposed conformational changes. We suggest that the model-structure and predicted motion represent alkaline pH-induced conformational changes, mediated by a cluster of evolutionarily conserved, titratable residues, at the cytoplasmic ends of TMs II, V, and IX.     

Phosphatidylinositol-specific phospholipase C-gamma1 undergoes pH-induced activation and conformational change

Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.     

Site-directed antibodies against the stem region reveal low pH-induced conformational changes of the Semliki Forest virus fusion protein

The E1 envelope protein of the alphavirus Semliki Forest virus (SFV) is a class II fusion protein that mediates low pH-triggered membrane fusion during virus infection. Like other class I and class II fusion proteins, during fusion E1 inserts into the target membrane and rearranges to form a trimeric hairpin structure. The postfusion structures of the alphavirus and flavivirus fusion proteins suggest that the "stem" region connecting the fusion protein domain III to the transmembrane domain interacts along the trimer core during the low pH-induced conformational change. However, the location of the E1 stem in the SFV particle and its rearrangement and functional importance during fusion are not known. We developed site-directed polyclonal antibodies to the N- or C-terminal regions of the SFV E1 stem and used them to study the stem during fusion. The E1 stem was hidden on neutral pH virus but became accessible after low pH-triggered dissociation of the E2/E1 heterodimer. The stem packed onto the trimer core in the postfusion conformation and became inaccessible to antibody binding. Generation of the E1 homotrimer on fusion-incompetent membranes identified an intermediate conformation in which domain III had folded back but stem packing was incomplete. Our data suggest that E1 hairpin formation occurs by the sequential packing of domain III and the stem onto the trimer core and indicate a tight correlation between stem packing and membrane merger.     

pH-induced conformational change of the influenza M2 protein C-terminal domain

The M2 protein from influenza A is a pH-activated proton channel that plays an essential role in the viral life cycle and serves as a drug target. Using spin labeling EPR spectroscopy, we studied a 38-residue M2 peptide spanning the transmembrane region and its C-terminal extension. We obtained residue-specific environmental parameters under both high- and low-pH conditions for nine consecutive C-terminal sites. The region forms a membrane surface helix at both high and low pH, although the arrangement of the monomers within the tetramer changes with pH. Both electrophysiology and EPR data point to a critical role for residue Lys 49.     

Characterizing DNA condensation and conformational changes in organic solvents

Organic solvents offer a new approach to formulate DNA into novel structures suitable for gene delivery. In this study, we examined the in situ behavior of DNA in N, N-dimethylformamide (DMF) at low concentration via laser light scattering (LLS), TEM, UV absorbance and Zeta potential analysis. Results revealed that, in DMF, a 21bp oligonucleotide remained intact, while calf thymus DNA and supercoiled plasmid DNA were condensed and denatured. During condensation and denaturation, the size was decreased by a factor of 8-10, with calf thymus DNA forming spherical globules while plasmid DNA exhibited a toroid-like conformation. In the condensed state, DNA molecules were still able to release the counterions to be negatively charged, indicating that the condensation was mainly driven by the excluded volume interactions. The condensation induced by DMF was reversible for plasmid DNA but not for calf thymus DNA. When plasmid DNA was removed from DMF and resuspended in an aqueous solution, the DNA was quickly regained a double stranded configuration. These findings provide further insight into the behavior and condensation mechanism of DNA in an organic solvent and may aid in developing more efficient non-viral gene delivery systems.