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PubMed1996

High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation: proton equilibrium, cations, and electrostatics.

The kinetics of pulsed-light photoactivation, the light-induced reassembly of the water-oxidizing complex (WOC) of PSII in the presence of essential inorganic cofactors, has been studied using two improvements: a new efficient chelator, N,N,N',N'-tetrapropionato-1,3-bis(aminomethyl)benzene (TPDBA), for complete extraction of {Mn4} and Ca2+ and an ultrasensitive polarographic cell for O2 detection [Ananyev, G.M., & Dismukes, G.C. (1996) Biochemistry 35, 4102-4109]. Measurements have been made of the initial half-time, t1/2 (sum of the lag time for formation of the first intermediate, IM1, plus the half-time for formation of the second intermediate, IM2), and the steady-state yield, Yss, for recovery of O2 evolution (proportional to the number of active centers). The following conclusions have been reached: (1) cations (Ca2+, Mg2+, and Na+) slow the rate of photoactivation, even though Ca2+ is essential for activity. Two distinct mechanisms appear to be involved: binding to one or both of the first two Mn(2+)-specific sites and screening of negative charges on apo-WOC that are responsible for concentrating Mn2+ ions by electrostatic steering; (2) the Michaelis constant for the calcium requirement for Yss at sufficiently low Mn2+ concentrations (8 microM) that competition at the calcium site does not occur is K(m) = 1.4 mM. Numerically, K(m) is the same for reactivation of O2 evolution in Ca-depleted PSII membranes which retain four Mn ions; (3) in the absence of Ca2+ but in the presence of saturating amounts of Mn2+ (8 Mn/apo-WOC) and Cl-(35 mM) assembly of a stable tetra-Mn cluster occurs neither under illumination nor in the dark after subsequent addition of CaCl2. However, in the presence of suboptimal concentrations of calcium required for maximum Yss, calcium-dependent assembly of stable yet inactive clusters occurs in the light; (4) protons in equilibrium with the buffer greatly increase the half-time 3-fold between pH 6.75 and 5.4, indicating ionization of one or more protons from the first photo-oxidized intermediate formed prior to the rate-limiting step (photo-oxidation of the second Mn2+); (5) the lipophilic membrane soluble anion tetraphenylboron (TPB-), a known reductant of intact WOC, increases the half-time 2.5-fold (< or = 40 microM) and paradoxically stimulates Yss by 50% at 20 microM concentration. These results suggest that TPB- increases the local concentration of Mn2+ adjacent to apo-WOC (Yss increase), while also reducing the S2 and S3 states of the intact WOC at higher concentrations (t1/2 increase). The effects of anions and cations indicates that overcoming the surface potential of the membrane/protein PSII complex may play an important role in the kinetics of reassembly of the {Mn4} cluster; (6) the ratio Y4/Y3 in the kinetics of O2 evolution from a series of single-turnover flashes, a ratio that typically reflects the probability of misses (alpha), grows noticeably larger with increasing extent of recovery of O2 evolving activity and also with increase in the amount of Mn2+, indicating competition between substrate water and excess Mn2+ for reduction of the functional {Mn4} cluster. On the basis of these results, we extend the model for photoactivation to include the antagonistic effects of H+ and Ca2+ in the formation of the first two intermediates.

Benzylamines↗
PubMed1996

Electrostatic interactions in the active site of the N-terminal thioredoxin-like domain of protein disulfide isomerase.

Proteins with the thioredoxin fold have widely differing stabilities of the disulfide bond that can be formed between the two cysteines at their active site sequence motif Cys1-Xaa2-Yaa3-Cys4. This is believed to be regulated not by varying the disulfide bond itself, but by modulating the stability of the dithiol form of the protein through interactions with the ionized form of the Cys1 thiol group. A consistent relationship between disulfide bond stability and Cys1 thiol pKa value is found here for DsbA, thioredoxin, and the N-terminal thioredoxin-like domain of protein disulfide isomerase (PDI a), which has a very low thiol pKa value of 4.5. This thiolate anion is stabilized by 5.7 kcal/mol in the dithiol form, giving rise to the corresponding instability of the disulfide bond and the oxidizing properties of PDI a. Electrostatic interactions in the active site of the PDI a-domain have been characterized in order to understand the physical basis of this stabilization. Linkage with the ionization of the imidazole group of His3 in the active site demonstrates that this charge-charge interaction contributes 1.1 kcal/mol. The remainder of the stabilization is believed to be due primarily to interactions with the partial positive charges at the N-terminus of an alpha-helix, which are exceedingly sensitive to charges of surrounding residues.

Binding Sites↗
PubMed1997

Electrostatic influence of QA reduction on the IR vibrational mode of the 10a-ester C==O of HA demonstrated by mutations at residues Glu L104 and Trp L100 in reaction centers from Rhodobacter sphaeroides.

The light-induced QA-/QA FTIR difference spectrum of the photoreduction of the primary quinone (QA) in reaction centers (RCs) from Rhodobacter sphaeroides exhibits a set of complex differential bands between 1750 and 1715 cm(-1). Several of these features correspond in frequency to bands that bleach in the HA-/HA FTIR difference spectra of the photoreduction of the bacteriopheophytin electron acceptor (HA). Since the 10a-ester C==O from HA and the side chains of protonated carboxylic acids would be expected to contribute in this spectral region, mutations were designed at Trp L100 and Glu L104, which have been proposed to form hydrogen bonds to the 10a-ester and the 9-keto carbonyls on ring V of HA, respectively. The QA/-/QA spectra measured in IH2O and 2H2O of RCs from wild type (WT) were compared to those of RCs with the mutation Trp to Phe at L100 [WF(L100)], Glu to Leu at L104 [EL(L104)], or both mutations [EL(L104)/WF(L100)]. The spectra of the mutants in the 1800-1400 cm(-1) frequency range exhibit only limited perturbations compared to those of WT, indicating the absence of significant structural changes due to the mutations. Part of a differential signal centered around 1732 cm(-1) in the spectrum of WT RCs is downshifted by approximately 7 cm(-1) in EL(L104), while it is upshifted by approximately 11 cm(-1) in WF(L100). This upshift of the differential signal is assigned to the frequency change of the 10a-ester C==O of HA induced by the rupture of the hydrogen bond with Trp L100. The 1H2O-minus-2H2O double-difference spectrum of WT RCs exhibits a characteristic differential signal positive at 1730 cm-1 and negative at 1724 cm-1 that is absent in the corresponding spectra of EL(L104) and of the double mutant, implicating Glu L104 in the QA-/QA spectral changes. This differential signal is strongly modified in frequency and amplitude in the 1H2O-minus-2H2O spectrum of WF(L100), indicating that it does not correspond to a direct response of the C==O mode of the Glu L104 side chain upon QA reduction. Instead, perturbation of the hydrogen bond of the 9-keto C==O with Glu L104 is proposed to induce a change of electron density on ring V of HA, thereby altering the frequency of the 10a-ester C==O that is in partial conjugation with ring V. The loss of the hydrogen bond to the 9-keto C==O of HA due to the Glu L104 to Leu mutation or the alteration of the strength of the hydrogen bond by 1H/2H exchange on Glu L104 appears to produce such effects. Thus, the QA-/QA spectra above 1700 cm-1 are dominated by contributions from the 10a-ester C==O of HA, with most of the differential signals assigned to a small frequency downshift of the 10a-ester C==O of HA in response to QA reduction. The complexity of the signals implies a structural heterogeneity of the conformation and hydrogen bonding of the 10a-ester C==O of HA, which may be related to the functional heterogeneity observed in electron transfer kinetics. The present FTIR results show that the reduction of QA can induce a pronounced electrostatic effect on molecular vibrations of chemical groups located about 10 A away from QA. They also demonstrate that, within experimental limits, the proton uptake observed at pH 7 upon QA photoreduction [McPherson, P. H., Okamura, M. Y., & Feher, G. (1988) Biochim. Biophys. Acta 934, 348-368] involves none of the exchangeable carboxylic groups of the RC.

Amino Acid Sequence↗
PubMed1997

Contribution of surface histidyl residues in the alpha-chain to the Bohr effect of human normal adult hemoglobin: roles of global electrostatic effects.

We have applied site-directed mutagenesis to our Escherichia coli hemoglobin expression plasmid and constructed five recombinant mutant hemoglobins (r Hbs): r Hb(alpha20His-->Gln or alpha:H20Q); r Hb(alpha:H50Q); r Hb(alpha:H72Q); r Hb(alpha:H89Q); and r Hb(alpha:H112Q). We have constructed these r Hbs to help us assess the contribution of the surface histidyl residues in the alpha-chain to the alkaline Bohr effect of human normal adult hemoglobin (Hb A). In our laboratory, we have monitored the variation of proton nuclear magnetic resonances arising from the C2 protons of the histidyl residues of Hb A as a function of pH and buffer conditions. Several of these resonances have been assigned to the individual histidyl residues on the surface of the hemoglobin molecule using naturally occurring mutant hemoglobins and chemically modified hemoglobins. In the present work, we have identified the C2 proton resonances of five surface histidyl residues of the alpha-chain, alpha20, alpha50, alpha72, alpha89, and alpha112, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance spectra of Hb A with those of the r Hbs. For the assignment of the C2 proton resonances of alpha20His and alpha112His, we have used combinations of mutations to compensate for the spectral perturbations resulting from the single mutations, which obscure the resonance assignment. On the basis of the new findings, in solvent containing 0.1 M chloride, the overall contributions from surface histidyl residues of both the alpha- and beta-chain and from other previously identified alkaline Bohr groups account for approximately 75% of the observed Bohr effect at pH 7.3 (the maximum Bohr effect under the prescribed solvent conditions). Our results show that some histidyl residues contribute to the Bohr effect and some oppose the net Bohr effect. In some cases, the addition of anions can diminish or reverse the contributions of specific histidyl residues to the overall Bohr effect. Thus, the Bohr effect, a heterotropic effect, depends on the intricate arrangement and interactions of all hydrogen and anion binding sites in the hemoglobin molecule. It is an excellent example of global electrostatic effects in proteins.

Adult↗
PubMed1997

A classical enzyme active center motif lacks catalytic competence until modulated electrostatically.

The cysteine proteinase superfamily is a source of natural structural variants of value in the investigation of mechanism. It has long been considered axiomatic that catalytic competence of these enzymes mirrors the generation of the ubiquitous catalytic site imidazolium-thiolate ion pair. We here report definitive evidence from kinetic studies supported by electrostatic potential calculations, however, that at least for some of these enzymes the ion pair state which provides the nucleophilic and acid-base chemistry is essentially fully developed at low pH where the enzymes are inactive. Catalytic competence requires an additional protonic dissociation with a common pKa value close to 4 possibly from the Glu50 cluster to control ion pair geometry. The pH dependence of the second-order rate constant (k) for the reactions of the catalytic site thiol groups with 4,4'-dipyrimidyl disulfide is shown to provide the pKa values for the formation and deprotonation of the (Cys)-S-/(His)-Im+H ion pair state. Analogous study of the reactions with 2,2'-dipyridyl disulfide reveals other kinetically influential ionizations, and all of these pKa values are compared with those observed in the pH dependence of kcat/Km for the catalyzed hydrolysis of N-acetylphenylalanylglycine 4-nitroanilide. The discrepancy between the pKa value for ion pair formation and the common pKa value close to 4 related to generation of catalytic activity is particularly marked for ficin (pKa 2.49 +/- 0.02) and caricain (pKa 2.88 +/- 0.02) but exists also for papain (pKa 3.32 +/- 0.01).

Binding Sites↗
PubMed1997

Electrostatic effects on substrate activation in para-hydroxybenzoate hydroxylase: studies of the mutant lysine 297 methionine.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein monooxygenase that catalyzes the incorporation of one atom of molecular oxygen into p-hydroxybenzoate to form 3,4-dihydroxybenzoate. The enzyme activates the substrate at the 3 position to electrophilic substitution by lowering the pKa of the phenolic oxygen. The results presented here indicate that regions of positive potential in the active site facilitate this substrate activation, which is necessary for rapid hydroxylation. We have neutralized a positive point charge by mutating lysine 297 to methionine (K297M). This mutation changes an amino acid near the active site, but not directly in contact with the flavin or the substrate. A variety of transient state kinetic and static parameters have been determined with two substrates. The results indicate that the K297M mutant does not activate the substrate through phenolic ionization to the same extent as wild-type (WT) and yet remains a competent hydroxylase. However, catalysis by the mutant is slow compared to that of WT, particularly in the oxidative half-reaction. Thus, normally quite labile oxygenated flavin intermediates encountered in the hydroxylation pathway of WT p-hydroxybenzoate hydroxylase are stabilized and their decay is rate limiting in the K297M turnover. Electrostatic potential calculations offer an explanation for the lack of substrate activation. The stability of the oxidative reaction intermediates seems to be related to a lower degree of substrate activation.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1997

Opposite effects of electrostatics and steric exclusion on bundle formation by F-actin and other filamentous polyelectrolytes.

A number of positively charged polypeptides and proteins bundle DNA, F-actin, microtubules, and viruses such as filamentous phage fd and tobacco mosaic virus (TMV), as well as intermediate filaments formed by vimentin. The general behavior is dictated by the common polyelectrolyte nature of these biopolymers, which gives rise to nonspecific binding by ligands carrying several net opposite charges. An attractive interaction accounts for the subsequent lateral aggregation, distinguishing this transition from the liquid crystalline formation of filamentous particles at high concentrations. Morphologically similar filament bundles can also be induced by inert solutes such as polyethylene glycol (PEG) and proteins that do not bind the macromolecular filaments, but the physicochemistry underlying this class of bundle transitions is distinct. In particular, bundling transitions induced by electrostatic and steric mechanisms have an opposite dependence on the solution ionic strength and the concentration of the filamentous biopolymers. The distinct mechanisms illustrated in this report may each contribute to the formation of specific polymer bundles under physiological conditions.

Actins↗
PubMed1998

Function of tyrosine Z in water oxidation by photosystem II: electrostatical promotor instead of hydrogen abstractor.

Photosynthetic water oxidation by photosystem II is mediated by a Mn4 cluster, a cofactor X still chemically ill-defined, and a tyrosine, YZ (D1-Tyr161). Before the final reaction with water proceeds to yield O2 (transition S4-->S0), two oxidizing equivalents are stored on Mn4 (S0-->S1-->S2), a third on X (S2-->S3), and a forth on YZ(S3-->S4). It has been proposed that YZ functions as a pure electron transmitter between Mn4X and P680, or, more recently, that it acts as an abstractor of hydrogen from bound water. We scrutinized the coupling of electron and proton transfer during the oxidation of YZ in PSII core particles with intact or impaired oxygen-evolving capacity. The rates of electron transfer to P680+, of electrochromism, and of pH transients were determined as a function of the pH, the temperature, and the H/D ratio. In oxygen-evolving material, we found only evidence for electrostatically induced proton release from peripheral amino acid residues but not from YZox itself. The positive charge stayed near YZox, and the rate of electron transfer was nearly independent of the pH. In core particles with an impaired Mn4 cluster, on the other hand, the rate of the electron transfer became strictly dependent on the protonation state of a single base (pK approximately 7). At pH < 7, the rate of electron transfer revealed the same slow rate (t1/2 approximately 35 microseconds) as that of proton release into the bulk. The deposition of a positive charge around YZox was no longer detected. A large H/D isotope effect (approximately 2.5) on these rates was also indicative of a steering of electron abstraction by proton transfer. That YZox was deprotonated into the bulk in inactive but not in oxygen-evolving material argues against the proposed role of YZox as an acceptor of hydrogen from water. Instead, the positive charge in its vicinity may shift the equilibrium from bound water to bound peroxide upon S3-->S4 as a prerequisite for the formation of oxygen upon S4-->S0.

Chlorophyll↗
PubMed1998

Calculations of electrostatic interactions and pKas in the active site of Escherichia coli thioredoxin.

The active-site protonation state is crucial to the reductive mechanism of Escherichia coli thioredoxin, which involves a nucleophilic attack by the thiolate form of Cys32. We have calculated the titration properties of the active-site residues using a continuum electrostatic model, the X-ray structure of the oxidized protein, and ensembles of NMR structures of the oxidized and reduced protein. Protein dipoles, especially the SH dipole of Cys35, can provide sufficient stabilization of the Cys32 thiolate to account for its low experimental pKa (approximately 7.4), but this effect is very sensitive to local conformational variations. The experimental finding that Cys32 titrates at a lower pH than Cys35 is explained by the latter's deeper burial from solvent exposure, and stronger interaction with the carboxylate of Asp26, and not by helix dipoles or positively charged side chains. The calculated very strong interaction between Cys32 and Cys35 in their thiolate forms implies that their titration must occur in two widely pH-separated steps and that the thiolate groups must move apart in the second step. The calculations are very consistent with the experimental Asp26 pKa value of 7.5 for the oxidized X-ray structure. Both the oxidized and reduced NMR structures fall into two categories: "tight" structures in which the Asp26 and Lys57 side chains are in direct contact, and for which the calculations predict unreasonably low pKas; and "loose" structures, which resemble the oxidized X-ray structure in that these side chains are farther apart, and for which the calculations are in very good agreement with experiment. We propose that the calculations over the NMR ensemble can be used as a test of the alternative structural models provided by NMR.

Aspartic Acid↗
PubMed1998

Electrostatic interactions at the donor side of the photosynthetic reaction center of Rhodopseudomonas viridis.

The photosynthetic reaction center of Rhodopseudomonas viridis, a purple bacterium, contains a tetraheme cytochrome subunit. After its photoinduced oxidation, the primary donor, P, is reduced by the nearby heme (c559) of the tetraheme subunit in about 200 ns. This heme, in turn, is reduced by another heme (c556) of the subunit in about 2 micro(s). The midpoint potentials of P, c559, and c556 are known to be +500, +380, and +320 mV, respectively. The reduction kinetics of P+ are strongly biphasic in living cells, membrane fragments, and isolated reaction centers. We show here that this biphasicity reflects a small equilibrium constant (lower than 10) for the electron-transfer reaction between P and c559, which arises from a significant difference between the operating redox potentials of the P+/P and c559+/c559 couples and their equilibrium midpoint potentials. This difference is partly due to the effect of the permanent transmembrane potential, which arises from the cell metabolism, and to significant electrostatic interactions which develop between the electron carriers of the reaction center. Interestingly, the kinetic parameters of P+ reduction in decoupled cells or membrane fragments are identical to those reported for isolated reaction centers. We estimate an interaction of about 20 mV between c556 and c559 and about 90 mV between c559 and P. Consequently, the operating redox potential of the P+/P couple is 410 mV.

Electron Transport↗
PubMed1998

Characterization of pKa values and titration shifts in the cytotoxic ribonuclease alpha-sarcin by NMR. Relationship between electrostatic interactions, structure, and catalytic function.

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, approximately 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.

Aspergillus↗
PubMed1998

Structure of the potassium form of CGCGAATTCGCG: DNA deformation by electrostatic collapse around inorganic cations.

The potassium form of d(CGCGAATTCGCG) solved by X-ray diffraction to 1.75 A resolution indicates that monovalent cations penetrate the primary and secondary layers of the "spine of hydration". Both the sodium [Shui, X., McFail-Isom, L., Hu, G. G., and Williams, L. D. (1998) Biochemistry 37, 8341-8355] and the potassium forms of the dodecamer at high resolution indicate that the original description of the spine, only two layers deep and with full occupancy by water molecules, requires substantive revision. The spine is merely the bottom two layers of a four layer solvent structure. The four layers combine to form a repeating motif of fused hexagons. The top two solvent layers were not apparent from previous medium-resolution diffraction data. We propose that the narrow minor groove and axial curvature of A-tract DNA arise from localization of cations within the minor groove. In general, the results described here support a model in which most or all forces that drive DNA away from canonical B-conformation are extrinsic and arise from interaction of DNA with its environment. Intrinsic forces, originating from direct base-base interactions such as stacking, hydrogen bonding, and steric repulsion among exocyclic groups appear to be insignificant. The time-averaged positions of the ubiquitous inorganic cations that surround DNA are influenced by DNA bases. The distribution of cations depends on sequence. Regions of high and low cation density are generated spontaneously in the solvent region by heterogeneous sequence or even within the grooves of homopolymers. The regions of high and low cation density deform DNA by electrostatic collapse. Thus, the effects of small inorganic cations on DNA structure are similar to the effects of proteins.

Cations, Monovalent↗
PubMed1999

pKa values and the pH dependent stability of the N-terminal domain of L9 as probes of electrostatic interactions in the denatured state. Differentiation between local and nonlocal interactions.

pKa values were measured for the 6 carboxylates in the N-terminal domain of L9 (NTL9) by following NMR chemical shifts as a function of pH. The contribution of each carboxylate to the pH dependent stability of NTL9 was estimated by comparing the pKa values for the native and denatured state of the protein. A set of peptides with sequences derived from NTL9 were used to model the denatured state. In the protein fragments, the pKa values measured for the aspartates varied between 3.8 and 4.1 and the pKa values measured for the glutamates varied between 4.1 and 4.6. These results indicate that the local sequence can significantly influence pKa values in the denatured state and highlight the difficulties in using standard pKa values derived from small compounds. Calculations based on the measured pKa values suggest that the free energy of unfolding of NTL9 should decrease by 4.4 kcal mol-1 when the pH is lowered from 6 to 2. In contrast, urea and thermal denaturation experiments indicate that the stability of the protein decreases by only 2.6 kcal mol-1 when the carboxylates are protonated. This discrepancy indicates that the protein fragments are not a complete representation of the denatured state and that nonlocal sequence effects perturb the pKa's in the denatured state. Increasing the salt concentration from 100 to 750 mM NaCl removes the discrepancy between the stabilities derived from denaturation experiments and the stability changes calculated from the pKa values. At high concentrations of salt there is also less variation of the pKa values measured in the protein fragments. Our results argue that in the denatured state of NTL9 there are electrostatic interactions between groups both local and nonlocal in primary sequence.

Amino Acid Sequence↗
PubMed1999

Electrostatic and aromatic microdomains within the binding-site crevice of the D2 receptor: contributions of the second membrane-spanning segment.

The binding-site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted cysteine accessibility method (SCAM), we previously mapped the residues in the third, fifth, sixth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the second membrane-spanning segment (M2) and expressed the mutant receptors in HEK 293 cells. Eleven of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and 9 of these 11 were protected from reaction by a reversible dopamine antagonist, sulpiride. We infer that the side chains of the residues at the 11 reactive loci (D80, L81, V83, V87, P89, W90, V91, V92, L94, E95, V96) are on the water-accessible surface of the binding-site crevice and that 9 of these are occluded by bound antagonist. The pattern of accessibility suggests an alpha-helical conformation. A broadening of the angle of accessibility near the binding site is consistent with the presence of a kink at Pro89. On the basis of the enhanced rates of reaction of positively charged sulfhydryl reagents, we infer the presence of an electrostatic microdomain composed of three acidic residues in M2 and the adjacent M3 that could attract and orient cationic ligands. Furthermore, based on the enhanced reactivity of the hydrophobic cation-containing reagent, we infer the presence of an aromatic microdomain formed between M2, M3, and M7.

Amino Acid Substitution↗
PubMed1999

8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions.

Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting partially folded states of proteins. We have carried out a systematic investigation on the effect of ANS, a charged hydrophobic fluorescent dye, on structural properties of acid-unfolded horse heart cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectroscopy (ESI MS). ANS was found to induce, a secondary structure similar to native protein and quenching of fluorescence of tryptophan residue, in the acid-unfolded protein. However, the tertiary structure was found to be disrupted thus indicating that ANS stabilizes a molten globule state in acid-unfolded protein. To understand the mechanism of ANS-induced folding of acid-unfolded cytochrome c, comparative ESI MS, soret absorption, and tryptophan fluorescence studies using nile red, a neutral hydrophobic dye, and ANS were carried out. These studies suggested that, at low pH, electrostatic interactions between negatively charged ANS molecules and positively charged amino acid residues present in acid-unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compact state or molten globule state. This is the first experimental demonstration of ANS induced folding of unfolded protein and puts to question the usefulness of ANS for characterization/determination of partially folded intermediates of proteins observed under low pH conditions.

Anilino Naphthalenesulfonates↗
PubMed1999

Formation of domains of cationic or anionic lipids in binary lipid mixtures increases the electrostatic coupling strength of water-soluble proteins to supported bilayers.

The electrostatic binding strength of water-soluble proteins having either an excess positive (cytochrome c) or negative (beta-lactoglobulin) electric charge to oppositely charged supported planar bilayers (SPBs) was studied as a function of the bilayer phase state (fluid or gel phase) by IR-ATR spectroscopy. The bilayer consisted of mixtures of zwitterionic DEPC with either cationic DMTAP or anionic DMPG. We observed drastic differences in the binding strength of both proteins for the two bilayer phase states, with the gel phase exhibiting a higher binding strength than the fluid phase, under conditions where the two lipid components had different hydrophobic chain lengths resulting in a nonideal mixing behavior. In addition, for beta-lactoglobulin we observed a strong binding to a gel phase SPB comprising DEPC/DMTAP, while raising the temperature of the SPB above the chain melting transition temperature of the mixture resulted in a complete unbinding of the protein. In contrast, for DMPC/DMTAP having the same cationic charge content but no hydrophobic chain mismatch, no phase-dependent coupling strength of the protein to the SPB was observed. Our results suggest that the formation of charge-enriched domains by partial demixing of the bilayer lipids at the transition to the gel state is crucial for modulation of the protein binding strength to the SPB, while the intrinsic charge of the solid support surface is of minor importance.

Animals↗
PubMed2000

Tryptophan-heme pi-electrostatic interactions in cytochrome f of oxygenic photosynthesis.

Cytochrome f of oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures of the chloroplast and cyanobacterial cytochromes f were compared to explain spectral and redox differences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis of cytochrome f from the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 --> Trp substitution in the N-terminal domain was unique in causing a red shift of 1 and 2 nm in the cytochrome Soret (gamma) and Q (alpha) visible absorption bands, respectively. The resulting alpha band peak at 556 nm is characteristic of the cyanobacterial cytochrome. Conversely, a Trp4 --> Phe mutation in the expressed cytochrome from the cyanobacterium Phormidium laminosum caused a blue shift to the 554 nm alpha band peak diagnostic of the chloroplast cytochrome. Residue 4 was found to be the sole determinant of this 60 cm(-)(1) spectral shift, and of approximately one-half of the 70 mV redox potential difference between cytochrome f of P. laminosum and C. reinhardtii (E(m7) = 297 and 370 mV, respectively). The proximity of Trp-4 to the heme implies that the spectral and redox potential shifts arise through differential interaction of its sigma- or pi-electrostatic potential with the heme ring and of the pi-potential with the heme Fe orbitals, respectively. The dependence of the visible spectrum and redox potential of cytochrome f on the identity of aromatic residue 4 provides an example of the use of the relatively sharp cytochrome spectrum as a "spectral fingerprint", and of the novel structural connection between the heme and a single nonliganding residue.

Amino Acid Sequence↗
PubMed2001

Assembly of alternating polycation and DNA multilayer films by electrostatic layer-by-layer adsorption.

The assembly of alternating DNA and positively charged poly(dimethyldiallylammonium chloride) (PDDA) multilayer films by electrostatic layer-by-layer adsorption has been studied. The real-time surface plasmon resonance (BIAcore) technique was used to characterize and monitor the formation of multilayer films in solution in real time continuously. Electrochemical impedance spectroscopy (EIS) and UV-vis absorbance measurements were also used to study the film assembly, and linear film growth was observed. All the results indicate that the uniform multilayer can be obtained on the poly(ethylenimine)- (PEI-) coated substrate surface. The kinetics of the adsorption of DNA on PDDA surface was also studied by the real-time BIAcore technique; the observed rate constant was calculated using a Langmuir model (k(obs) = (1.28 +/- 0.08) x 10(-2) s(-1).

Adsorption↗