Domain formation in phosphatidylinositol monophosphate/phosphatidylcholine mixed vesicles

Phosphoinositides have been shown to control membrane trafficking events by targeting proteins to specific cellular sites, which requires a tight regulation of phosphoinositide generation and turnover as well as a high degree of compartmentalization. To shed light on the processes that lead to the formation of phosphoinositide-enriched microdomains, phosphatidylcholine/phosphatidylinositol monophosphate (phosphatidylinositol-3-phosphate (PI-3P), -4-phosphate (PI-4P), or -5-phosphate (PI-5P)) mixed vesicles were investigated by calorimetric (DSC) Fourier transform infrared spectroscopic (FTIR), and fluorescence resonance energy transfer (FRET) measurements. The experiments furnished results consistent with a pH-dependent formation of phosphatidylinositol monophosphate-enriched microdomains. The domain formation was most pronounced between pH approximately 7 and approximately 9.5, whereas slightly acidic pH values (pH 4) resulted in the disintegration of the domains. This pH-dependent phosphatidylcholine/phosphatidylinositol monophosphate demixing was observed for the gel phase (FTIR experiments) as well as for the fluid lipid phase (FRET measurements). The observed microdomains are presumably stabilized by hydroxyl/hydroxyl as well as hydroxyl/phosphomonoester and phosphodiester interactions. While the pH dependence of the mutual phosphatidylinositol monophosphate interaction was largely the same for all investigated phosphatidylinositol monophosphates, it turned out that the relative stability of phosphatidylinositol monophosphate-enriched microdomains (pH 7-9.5) was governed by the position of the phosphomonoester group at the inositol ring (PI-4P > PI-5P > PI-3P). Demixing was also observed for phosphatidylcholine/phosphatidylinositol mixed vesicles; however, in this case the microdomain formation was only slightly affected by pH changes.     

Phosphatidylinositol-4,5-bisphosphate ionization and domain formation in the presence of lipids with hydrogen bond donor capabilities

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) is an important lipidic signaling molecule that is involved in a broad range of cellular processes. Its interaction with proteins and its lateral distribution are governed by the ionization state of the phosphomonoester groups and its ability to form intra- and intermolecular hydrogen bonds. In this study we have investigated the ionization state of PI(4,5)P(2) in ternary lipid vesicle systems that contain in addition to PI(4,5)P(2) and phosphatidylcholine (PC) either phosphatidylethanolamine (PE), phosphatidylserine (PS) or phosphatidylinositol (PI). In the presence of PE we find an increased ionization of PI(4,5)P(2), which can be attributed to increased deprotonation due to hydrogen bond formation between PE and the PI(4,5)P(2) phosphomonoester groups. However, the effect of PE on PI(4,5)P(2) ionization is significantly smaller than it had been found previously for phosphatidic acid in the presence of PE (Kooijman et al., 2005). The reduced impact of PE on PI(4,5)P(2) ionization can be attributed to competing intramolecular hydrogen bond formation between the phosphomonoester groups and neighboring hydroxyl groups. It is noteworthy that the presence of PE affects more strongly the ionization of the 5-phosphate group than that of the 4-phosphate, suggesting that the interaction of PE with the 5-phosphate is stronger. In PI(4,5)P(2)/PS/PC lipid vesicles, the presence of PS was expected to yield an increased protonation of the PI(4,5)P(2) phosphomonoester groups due to a decreased interfacial pH as a result of the increased negative interfacial charge. However, the effect of PS on PI(4,5)P(2) ionization is only minor, potentially suggesting that PS and PI(4,5)P(2) are demixed. The PI(4,5)P(2)/PI/PC vesicle system was characterized by a surprising mixing behavior that has potentially far reaching consequences: fluorescence microscopy measurements of giant unilammellar vesicles composed of PI(4,5)P(2)/PI/PC at physiological concentrations show that PI and PI(4,5)P(2) form macroscopic, fluid phase domains in contact with a fluid PC rich phase (fluid/fluid demixing). Despite the fact that PI and PI(4,5)P(2) co-localize, the effect of PI on PI(4,5)P(2) ionization behavior is only noticeable above pH 7. Apparently two opposing effects lead to the observed behavior: Due to the presence of the anionic PI, the interfacial pH drops, which is expected to lead to an enhanced protonation of the PI(4,5)P(2) phosphomonoester groups. In turn, hydrogen bond formation between PI and PI(4,5)P(2) would lead to the opposite, i.e. increased deprotonation of the phosphomonoester group. Apparently these two effects compensate each other for pH values smaller than about 7, while for higher pH values the increased interfacial pH in the presence of PI has a stronger impact than PI/PI(4,5)P(2) hydrogen bond formation. The cooperative formation of PI/PI(4,5)P(2) mixed domains has potentially important ramifications for the spatial organization of phosphoinositide mediated signaling events.     

Coexistence of two liquid crystalline phases in dihydrosphingomyelin and dioleoylphosphatidylcholine binary mixtures

Recently, DHSM, a minor constituent in naturally occurring SMs, was indicated to form a raft-like ordered phase more effectively than a naturally occurring form of SM because DHSM has greater potential to induce the intermolecular hydrogen bond. In order to examine the influence of the DHSM-induced hydrogen bond on the phase segregation, the thermal phase behavior of stearoyl-DHSM/DOPC binary bilayers was examined using calorimetry and fluorescence observation and compared with that of SSM/DOPC binary bilayers. Results revealed that the DHSM/DOPC bilayers undergo phase segregation between two L_α phases within a limited compositional range. On the other hand, apparent phase separation was not observed above main transition temperature in SSM/DOPC mixtures. Our monolayer measurements showed that the lipid packing of DHSM is less perturbed than that of SSM by the addition of small amount of DOPC, indicating a stronger hydrogen bond between DHSM molecules. Therefore, in DHSM/DOPC binary bilayers, DHSM molecules may locally accumulate to form a DHSM-rich domain due to a DHSM-induced hydrogen bond. On the other hand, excess accumulation of DHSM should be prevented because the difference in the curvature between DHSM and DOPC assemblies causes elastic constraint at the domain boundary between the DHSM-rich and DOPC-rich domains. Competition between the energetic advantages provided by formation of the hydrogen bond and the energetic disadvantage conferred by elastic constraints likely results in L_α/L_α phase separation within a limited compositional range.     

Characterization of the reversible conformational equilibrium in the cytoplasmic domain of human erythrocyte membrane band 3

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, and a tyrosine kinase, p72syk. cdb3 exists in a reversible, pH-dependent conformational equilibrium characterized by large changes in Stokes radius (11 A) and intrinsic fluorescence (2-fold). Based on the crystallographic structure of the cdb3 dimer, we hypothesized that the above conformational equilibrium might involve the movement of flanking peripheral protein binding domains away from a shared dimerization domain. To test this hypothesis, we have mutated both donor (W105L) and acceptor (D316A) residues of a prominent H bond that bridges the above two domains and have examined the effect on the resulting conformational equilibrium. Analysis of the intrinsic fluorescence, Stokes radius, thermal stability, urea stability, and segmental mobility of these mutants reveals that the above H bond is indeed present in the low pH conformation of cdb3 and broken in a higher pH conformation. The data further reveal that cdb3 exists in three native pH-dependent conformations and that rupture of the aforementioned H bond occurs only during conversion of the low pH conformation to the mid-pH conformation. Conversion of the mid-pH conformation to the high pH conformation would now appear to involve structural changes primarily in the peripheral protein binding domain. Because ankyrin associates avidly with the low pH conformation of cdb3, ankyrin occupancy should strongly influence this structural equilibrium and thereby affect band 3 and perhaps global membrane properties.     

Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K(+) channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (1)H(15)N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K(+) channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K(+) channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.     

Histidine-mediated pH-sensitive regulation of M-ficolin:GlcNAc binding activity in innate immunity examined by molecular dynamics simulations

Background

M-ficolin, a pathogen recognition molecule in the innate immune system, binds sugar residues including N-acetyl-D-glucosamine (GlcNAc), which is displayed on invading microbes and on apoptotic cells. The cis and trans Asp282-Cys283 peptide bond in the M-ficolin, which was found to occur at neutral and acidic pH in crystal structures, has been suggested to represent binding and non-binding activity, respectively. A detailed understanding of the pH-dependent conformational changes in M-ficolin and pH-mediated discrimination mechanism of GlcNAc-binding activity are crucial to both immune-surveillance and clearance of apoptotic cells.

Methodology/Principal Findings

By immunodetection analysis, we found that the pH-sensitive binding of GlcNAc is regulated by a conformational equilibrium between the active and inactive states of M-ficolin. We performed constant pH molecular dynamics (MD) simulation at a series of pH values to explore the pH effect on the cis-trans isomerization of the Asp282-Cys283 peptide bond in the M-ficolin fibrinogen-like domain (FBG). Analysis of the hydrogen bond occupancy of wild type FBG compared with three His mutants (H251A, H284A and H297A) corroborates that His284 is indispensible for pH-dependent binding. H251A formed new but weaker hydrogen bonds with GlcNAc. His297, unlike the other two His mutants, is more dependent on the solution pH and also contributes to cis-trans isomerization of the Asp282-Cys283 peptide bond in weak basic solution.

Conclusions/Significance

Constant pH MD simulation indicated that the cis active isomer of Asp282-Cys283 peptide bond was predominant around neutral pH while the trans bond gradually prevailed towards acidic environment. The protonation of His284 was found to be associated with the trans-to-cis isomerization of Asp282-Cys283 peptide bond which dominantly regulates the GlcNAc binding. Our MD simulation approach provides an insight into the pH-sensitive proteins and hence, ligand binding activity.     

The effect of catechol O-methylation on radical scavenging characteristics of quercetin and luteolin--a mechanistic insight

The biological effect of flavonoids can be modulated in  vivo due to metabolism. The O-methylation of the catechol group in the molecule by catechol O-methyl transferase is one of the important metabolic pathways of flavonoids. In the present study, the consequences of catechol O-methylation for the pH-dependent radical scavenging properties of quercetin and luteolin were characterized both experimentally and theoretically. Comparison of the pKa values to the pH-dependent TEAC profiles reveals that O-methylation not only affects the TEAC as such but also modulates the effect of changing pH on this radical scavenging activity due to an effect on the pKa for deprotonation. The pH-dependent TEAC curves and computer calculated electronic parameters: bond dissociation energy (BDE) and ionisation potential (IP) even indicate that O-methylation of the luteolin catechol group affects the radical scavenging potential only because it shifts the pKa for deprotonation. O-Methylation of the quercetin catechol moiety affects radical scavenging capacity by both an effect on the pKa, and also by an effect on the electron and hydrogen atom donating properties of the neutral (N) and the anionic (A) form of the molecule. Moreover, O-methylation of a catechol OH-group in quercetin and luteolin has a similar effect on their TEAC profiles and on calculated parameters as replacement of the OH-group by a hydrogen atom. Altogether, the results presented provide new mechanistic insight in the effect of catechol O-methylation on the radical scavenging characteristics of quercetin and luteolin.     

Probing electrostatic interactions along the reaction pathway of a glycoside hydrolase: histidine characterization by NMR spectroscopy

We have characterized by NMR spectroscopy the three active site (His80, His85, and His205) and two non-active site (His107 and His114) histidines in the 34 kDa catalytic domain of Cellulomonas fimi xylanase Cex in its apo, noncovalently aza-sugar-inhibited, and trapped glycosyl-enzyme intermediate states. Due to protection from hydrogen exchange, the level of which increased upon inhibition, the labile 1Hdelta1 and 1H epsilon1 atoms of four histidines (t1/2 approximately 0.1-300 s at 30 degrees C and pH approximately 7), as well as the nitrogen-bonded protons in the xylobio-imidazole and -isofagomine inhibitors, could be observed with chemical shifts between 10.2 and 17.6 ppm. The histidine pKa values and neutral tautomeric forms were determined from their pH-dependent 13C epsilon1-1H epsilon1 chemical shifts, combined with multiple-bond 1H delta2/epsilon1-15N delta1/epsilon2 scalar coupling patterns. Remarkably, these pKa values span more than 8 log units such that at the pH optimum of approximately 6 for Cex activity, His107 and His205 are positively charged (pKa > 10.4), His85 is neutral (pKa < 2.8), and both His80 (pKa = 7.9) and His114 (pKa = 8.1) are titrating between charged and neutral states. Furthermore, upon formation of the glycosyl-enzyme intermediate, the pKa value of His80 drops from 7.9 to <2.8, becoming neutral and accepting a hydrogen bond from an exocyclic oxygen of the bound sugar moiety. Changes in the pH-dependent activity of Cex due to mutation of His80 to an alanine confirm the importance of this interaction. The diverse ionization behaviors of the histidine residues are discussed in terms of their structural and functional roles in this model glycoside hydrolase.     

Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities.

Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4-(spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications.     

Catalytic function and local proton structure at the type 2 copper of nitrite reductase: the correlation of enzymatic pH dependence,conserved residues,and proton hyperfine structure

Electron nuclear double resonance (ENDOR) of protons at Type 2 and Type 1 cupric active sites correlates with the enzymatic pH dependence, the mutation of nearby conserved, nonligating residues, and electron transfer in heterologously expressed Rhodobacter sphaeroides nitrite reductase. Wild-type enzyme showed a pH 6 activity maximum but no kinetic deuterium isotope effect, suggesting protons are not transferred in the rate-limiting step of nitrite reduction. However, protonatable Asp129 and His287, both located near the Type 2 center, modulated enzyme activity. ENDOR of the wild-type Type 2 center at pH 6.0 revealed an exchangeable proton with large hyperfine coupling. Dipolar distance estimates indicated that this proton was 2.50-2.75 or 2.25-2.45 A from Type 2 copper in the presence or absence of nitrite, respectively. This proton may provide a properly oriented hydrogen bond to enhance water formation upon nitrite reduction. This proton was eliminated at pH 5.0 and showed a diminished coupling at pH 7.5. Mutations of Asp129 and His287 reduced enzyme activity and altered the exchangeable proton hyperfine spectra. Mutation of Asp129 prevented a pH-dependent change at the Type 1 Cys167 ligand as observed by Cys C(beta) proton ENDOR, implying there is a Type 2 and pH-dependent alteration of the Type 1 center. Mutation of the Type 1 center ligand Met182 to Thr and mutation of Asp129 increased the activation energy for nitrite reduction. Involvement of both the Type 1 center and Asp129 in modulating activation energy shows that electron transfer from the Type 1 center to a nitrite-ligated Type 2 center is rate-limiting for nitrite reduction. Mutation of Ile289 to Ala and Val caused minor perturbation to enzyme activity, but as detected by ENDOR, allowed formate binding. Thus, bulky Ile289 may exclude non-nitrite ligands from the Type 2 active site.     

Sequential unfolding of the two-domain protein Pseudomonas stutzeri cytochrome c(4)

P. stutzeri cytochrome c(4) is a di-haem protein, composed of two globular domains each with His-Met coordinated haem, and a hydrogen bond network between the domains. The domain foldings are highly symmetric but with specific differences including structural differences of ligand coordination, and different spin states of the oxidised haem groups. We have studied unfolding of oxidised P. stutzeri cyt c(4) induced thermally and by chemical denaturants. Horse heart cyt c was a reference molecule. Isothermal unfolding induced by guanidinium chloride and acid was followed by Soret, alpha/beta, and 701-nm band absorption, and by far-UV circular dichroism spectroscopy. Multifarious patterns emerge, but the two domains clearly unfold sequentially. One phase, assigned to unfolding of the N-terminal domain, proceeds at guanidinium concentrations up to approximately 1.0 M. This is followed by two overlapping phases at higher concentrations. The intermediate state maintains Fe-Met coordination, assigned to the C-terminal domain. Interdomain interaction is reflected in decreasing values of the cooperativity parameters. Differential scanning calorimetry shows a single peak, but two peaks appear when guanidinium chloride up to 0.4 M is present. This reflects different chemical action in chemical and thermal unfolding. Acid-induced unfolding kinetics was addressed by pH jumps using diode array stopped-flow techniques. Three kinetic phases in the 701 nm Fe-Met marker band, and four phases in the Soret and alpha/beta bands, spanning 4-1000 ms could be distinguished on pH jumps from 7.5 to the range 2.5-3.5. In this range of time and pH cyt c appears to unfold in no more than two phases. Spectral properties of the kinetic intermediates could be identified. Sequential domain unfolding, formation of high-spin states, and an intermediate state with Fe-Met coordination to a single haem group are features of the unfolding kinetics.     

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.     

pH-dependent conformational flexibility within the ribosomal peptidyl transferase center.

A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known.     

Angiotensin I-converting enzyme transition state stabilization by HIS1089: evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His(1089) as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His(1089) to Ala or Leu. At pH 7.5, with Ang I as substrate, k(cat)/K(m) values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (k(cat)) with minor effects on ground state substrate binding (K(m)). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His(1089) --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His(1089) side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His(1089) on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.     

Study of the swelling dynamics with overshooting effect of hydrogels based on sodium alginate-g-acrylic acid

A series of hydrogels were synthesized by graft cross-link copolymerization of sodium alginate (SA) and acrylic acid (AA) using N, N-methylene-bis-(acrylamide) as a cross-linker. By study of the swelling kinetics of the hydrogels in different buffer solutions, the overshooting effect was observed in acidic medium, namely the gels firstly swelled to a maximum value following by a gradual deswelling until the equilibrium. The phenomenon is interpreted as a cooperative physical cross-linking caused by the hydrogen bond formation between the carboxyl groups of the hydrogels in a hydrophobic environment. The hydrogen bond formation was further confirmed by FT-IR spectra. The dependence of overshooting effect on the pH of buffer solution was more noticeable in comparison with the composition of hydrogels, demonstrating that the cooperative physical cross-linking caused by the hydrogen bond formation is dominant. Whether or not the overshooting effect appears is not only relative to the pH of buffer solution, but also depends on the pK_a of carboxyl groups on the network. The overshoot processes of the hydrogels under acidic medium at pH below the pK_a follow a quantitative model proposed by Díez-Peńa et al., and the theoretical curves are in very good agreement with the experimental data. While in pH > pK_a buffer solutions, the overshoot phenomenon does not appear arising from the repulsive interaction between the ionized carboxyl groups, the swelling processes follow Schott second-order rate equation.     

Reduction potentials of Rieske clusters: importance of the coupling between oxidation state and histidine protonation state

Rieske [2Fe-2S] clusters can be classified into two groups, depending on their reduction potentials. Typical high-potential Rieske proteins have pH-dependent reduction potentials between +350 and +150 mV at pH 7, and low-potential Rieske proteins have pH-independent potentials of around -150 mV at pH 7. The pH dependence of the former group is attributed to coupled deprotonation of the two histidine ligands. Protein-film voltammetry has been used to compare three Rieske proteins: the high-potential Rieske proteins from Rhodobacter sphaeroides (RsRp) and Thermus thermophilus (TtRp) and the low-potential Rieske ferredoxin from Burkholderia sp. strain LB400 (BphF). RsRp and TtRp differ because there is a cluster to serine hydrogen bond in RsRp, which raises its potential by 140 mV. BphF lacks five hydrogen bonds to the cluster and an adjacent disulfide bond. Voltammetry measurements between pH 3 and 14 reveal that all the proteins, including BphF, have pH-dependent reduction potentials with remarkably similar overall profiles. Relative to RsRp and TtRp, the potential versus pH curve of BphF is shifted to lower potential and higher pH, and the pK(a) values of the histidine ligands of the oxidized and reduced cluster are closer together. Therefore, in addition to simple electrostatic effects on E and pK(a), the reduction potentials of Rieske clusters are determined by the degree of coupling between cluster oxidation state and histidine protonation state. Implications for the mechanism of quinol oxidation at the Q(O) site of the cytochrome bc(1) and b(6)f complexes are discussed.     

Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334-->Gly mutant enzymes

His334 facilitates catalysis by Corynebacterium callunae starch phosphorylase through selective stabilization of the transition state of the reaction, partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate. We have substituted His334 by a Gly and measured the disruptive effects of the site-directed replacement on active site function using steady-state kinetics and NMR spectroscopic characterization of the cofactor pyridoxal 5'-phosphate and binding of carbohydrate ligands. Purified H334G showed 0.05% and 1.3% of wild-type catalytic center activity for phosphorolysis of maltopentaose (kcatP = 0.033 s(-1)) and substrate binding affinity in the ternary complex with enzyme bound to phosphate (Km = 280 mm), respectively. The 31P chemical shift of pyridoxal 5'-phosphate in the wild-type was pH-dependent and not perturbed by binding of arsenate. At pH 7.25, it was not sensitive to the replacement His334-->Gly. Analysis of interactions of alpha-d-glucose 1-phosphate and alpha-d-xylose 1-phosphate upon binding to wild-type and H334G phosphorylase, derived from saturation transfer difference NMR experiments, suggested that disruption of enzyme-substrate interactions in H334G was strictly local, affecting the protein environment of sugar carbon 6. pH profiles of the phosphorolysis rate for wild-type and H334G were both bell-shaped, with the broad pH range of optimum activity in the wild-type (pH 6.5-7.5) being narrowed and markedly shifted to lower pH values in the mutant (pH 6.5-7.0). External imidazole partly restored the activity lost in the mutant, without, however, participating as an alternative nucleophile in the reaction. It caused displacement of the entire pH profile of H334G by + 0.5 pH units. A possible role for His334 in the formation of the oxocarbenium ion-like transition state is suggested, where the hydrogen bond between its side chain and the 6-hydroxyl polarizes and positions O-6 such that electron density in the reactive center is enhanced.     

Solid state NMR reveals a pH-dependent antiparallel beta-sheet registry in fibrils formed by a beta-amyloid peptide

We report solid state nuclear magnetic resonance (NMR) measurements that probe the supramolecular organization of beta-sheets in the cross-beta motif of amyloid fibrils formed by residues 11-25 of the beta-amyloid peptide associated with Alzheimer's disease (Abeta(11-25)). Fibrils were prepared at pH 7.4 and pH 2.4. The solid state NMR data indicate that the central hydrophobic segment of Abeta(11-25) (sequence LVFFA) adopts a beta-strand conformation and participates in antiparallel beta-sheets at both pH values, but that the registry of intermolecular hydrogen bonds is pH-dependent. Moreover, both registries determined for Abeta(11-25) fibrils are different from the hydrogen bond registry in the antiparallel beta-sheets of Abeta(16-22) fibrils at pH 7.4 determined in earlier solid state NMR studies. In all three cases, the hydrogen bond registry is highly ordered, with no detectable "registry-shift" defects. These results suggest that the supramolecular organization of beta-sheets in amyloid fibrils is determined by a sensitive balance of multiple side-chain-side-chain interactions. Recent structural models for Abeta(11-25) fibrils based on X-ray fiber diffraction data are inconsistent with the solid state NMR data at both pH values.     

Inorganic phosphate binding and electrostatic effects in the active center of aspartate aminotransferase apoenzyme.

The ionization state of the phosphate group bound at the aspartate aminotransferase apoenzyme's active site has been investigated utilizing Fourier-transform infrared spectroscopy following the band corresponding to the symmetric stretching of the dianionic phosphate. Unlike free phosphate, when inorganic phosphate is bound at the enzyme's active site, the integrated intensity value of the dianionic band does not change with pH within the studied range, and this value is similar to that for free dianionic phosphate at pH 8.3. From these results, we propose a dianionic state for the phosphate ion bound to cytosolic aspartate aminotransferase throughout the pH range of 5.7-8.3. The presence of other anions such as acetate and chloride or the substrate aspartate and its analogues produces a pH-dependent phosphate removal from the active site which is favored at low pH values. Elimination of the charged primary amine at the active-site Lys-258, through formation of a Schiff base with pyridoxal or chemical modification by carbamylation, also produces a pH-independent phosphate release. These results are interpreted as Lys-258 together with the active-site alpha-helix and other residues may be involved in stabilizing phosphate as a dianion in the apoenzyme phosphate pocket which anchors the phosphate ester of pyridoxal phosphate in the holoenzyme. It is proposed that the dianionic phosphate contributes to the apoenzyme's thermal stability through formation of strong hydrogen bond and salt bridges with the amino acid residues forming the phosphate binding pocket with assistance of Lys-258, and other active-site cationic components.     

Essential mechanisms in the catalysis of peptide bond formation on the ribosome

Peptide bond formation is the main catalytic function of the ribosome. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, amino-acyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.