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Electrostatic binding with tat and other cationic peptides increases cell accumulation of 99mTc-antisense DNAs without entrapment.

UNLABELLED: An important limitation restricting antisense nuclear medicine imaging is low radioactivity accumulations in target cells. The Tat peptide (Tat), a basic domain of the HIV Tat protein, has been shown to enhance cell accumulation of various biomolecules. PURPOSE: The influence of Tat, as a cationic carrier, on the accumulation in cell culture of anionic antisense DNAs bound electrostatically rather than covalently was investigated. To establish specificity of the accumulation, antisense DNA and control sequence were studied along with four different peptides. The technique of in situ reverse transcription was used to assay the in vivo hybridization of antisense DNA to the target mRNA in cultured live cells when transducted with the Tat peptide. METHODS: Uniform phosphorothioated DNAs were radiolabeled with 99mTc via Hynic/tricine. This 18 mer antisense DNA against RI alpha mRNA along with its sense and random control was studied in ACHN cells with the four peptides as carriers. RESULTS: The addition of Tat significantly increased cell accumulations. At 12 hours accumulations went from 14% to 45% for the antisense DNA and from 4% to 12% for control. Furthermore, an antisense effect was again suggested, now with the Tat carrier, by the significantly higher accumulation of 99mTc on both antisense DNAs vs. controls. Moreover, the accumulated antisense DNA enhanced with the Tat carrier was capable of priming reverse transcription as determined by an in situ assay suggesting that the DNA could escape from entrapment in endosome or lysosome vesicles for hybridization. However, differences in cellular accumulation with either Tat compared to either scrambled peptide were not significant, showing that the Tat in this study was functioning merely as a cationic carrier. CONCLUSIONS: Although electrostatic binding of antisense DNA to Tat is convenient, the association may mask the unique transduction properties of the peptide. Nevertheless, a promising improvement in cellular accumulation of antisense DNA was observed through the use of these carriers. In addition, at least a fraction of the transducted DNA appears to be free of entrapment to hybridize to its mRNA target in live cells.

DNA, Antisense↗
PubMed2001

A mechanism of separation in electrostatic ion chromatography.

The retention mechanism of electrostatic ion chromatography (EIC) is currently under debate and is the focus of this paper. A comprehensive set of retention data has been obtained on a C18 column coated with the zwitterionic surfactant 3-(N,N-dimethylmyristylammonio)propanesulfonate used with a range of mobile phases in which both the mobile-phase anion and cation have been varied systematically. Electro-osmotic flow measurements were also obtained on fused-silica capillaries coated with the zwitterion (and also some monofunctional surfactants) and were used to evaluate the nature of the surface charge on the layer of adsorbed surfactant in the presence of various background electrolytes. A new retention mechanism for EIC was developed on the basis of these data. This mechanism proposes that equilibration of the bound zwitterions with a mobile phase containing a suitable electrolyte causes the establishment of a charged layer created by the terminal sulfonate groups of the zwitterion, which acts as a Donnan membrane. The magnitude and polarity of the charge on this membrane depends on the nature of the mobile-phase ions. The Donnan membrane exerts weak electrostatic repulsion or attraction effects on analyte anions. A second component of the retention mechanism is chaotropic interaction of the analyte anion with the quaternary ammonium functional group of the zwitterion. This interaction exerts the major effect on the separation selectivity of EIC, such that analyte anions are eluted in order of increasing chaotropic interactions in accordance with the Hofmeister series.

Journal Article↗
PubMed2004

Combining mass spectrometry and pull-down techniques for the study of receptor heteromerization. Direct epitope-epitope electrostatic interactions between adenosine A2A and dopamine D2 receptors.

Previous results from FRET and BRET experiments and computational analysis (docking simulations) have suggested that a portion of the third intracellular loop (I3) of the human dopamine D2 receptor (D2R) and the C-tail from the human adenosine A2A receptor (A2AR) are involved in A2AR-D2R heteromerization. The results of the present studies, using pull-down and mass spectrometry experiments, suggest that A2AR-D2R heteromerization depends on an electrostatic interaction between an Arg-rich epitope from the I3 of the D2R (217RRRRKR222) and two adjacent Asp residues (DD401-402) or a phosphorylated Ser (S374) residue in the C-tail of the A2AR. A GST-fusion protein containing the C-terminal domain of the A2AR (GST-A2ACT) was able to pull down the whole D2R solubilized from D2R-tranfected HEK-293 cells. Second, a peptide corresponding to the Arg-rich I3 region of the D2R (215VLRRRRKRVN224) and bound to Sepharose was able to pull down both GST-A2ACT and the whole A2AR solubilized from A2AR-tranfected HEK-293 cells. Finally, mass spectometry and pull-down data showed that the Arg-rich D2R epitope binds to two different epitopes from the C-terminal part of the A2AR, containing the two adjacent Asp residues or the phosphorylated Ser residue (388HELKGVCPEPPGLDDPLAQDGAVGS412 and 370SAQEpSQGNT378). The present results are the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization, a new, expanding area of protein-protein interactions.

Amino Acid Sequence↗
PubMed2006

Electrochemical genosensor based on peptide nucleic acid-mediated PCR and asymmetric PCR techniques: Electrostatic interactions with a metal cation.

The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 degrees C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The attachment of long single-strands on GCE surface caused the accumulation of [Co(NH3)6]3+ and a high current response. Here, we report a versatile method that would allow for simple and rapid analysis of nucleic acids in combination with PNA-mediated PCR and asymmetric PCR techniques by using an electrochemical genosensor.

Base Sequence↗
PubMed2006

Electrostatic switching of biopolymer layers. Insights from combined electrokinetics and reflectometric interference.

Structural integrity and functional characteristics of biomacromolecules are largely defined by electrostatic forces between ionized moieties, which are often altered at interfaces. Unraveling these changes requires access to charge state and structure of surface-confined biopolymers in aqueous environments. We therefore combined electrokinetic measurements of interfacial electrical potentials with the simultaneous determination of the optical layer thickness by reflectometric interference spectroscopy. Two examples are summarized to demonstrate the resulting options: The pH-switching of grafted poly(l-glutamic acid) layers caused by dissociation-dependent helix-coil transitions was studied; potential distribution and ion mobility within the grafted polyelectrolyte were unraveled using an new theoretical model for the charging of polyelectrolyte layers. The charge-driven modulation of biopolymers at interfaces was furthermore analyzed in the adsorption of fibronectin onto polymer substrates with varied charge density; the results permit us to reach a conclusion about the relevance of electrostatic matching for orientation and anchorage of the protein. Altogether, the introduced methodology was found suitable to follow the electrosurface characteristics of biomacromolecules in situ.

Biopolymers↗
PubMed1995

Electrostatic effects of surface acidic amino acid residues on the oxidation-reduction potentials of the flavodoxin from Desulfovibrio vulgaris (Hildenborough).

The flavodoxin from Desulfovibrio vulgaris (Hildenborough) is a member of a family of small, acidic proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor. These proteins function as low-potential one-electron transferases in bacteria. A distinguishing feature of these flavoproteins is the dramatic decrease in the midpoint potential of the semiquinone/hydroquinone couple of the FMN upon binding to the apoprotein (-172 mV for FMN free in solution versus -443 mV when bound), a perturbation thought to be essential for physiological function. The structural basis of this phenomenon is not yet thoroughly understood. In this study, the contribution of six acidic residues (Asp62, Asp63, Glu66, Asp95, Glu99, and Asp106) to the perturbation of the redox properties of the cofactor has been investigated. These residues are clustered about the FMN binding site within 13 A of the N(1) atom of the cofactor. Using oligonucleotide-directed mutagenesis, these residues were neutralized in various combinations through the substitution of asparagine for aspartate and glutamine for glutamate. Seventeen mutant flavodoxins were generated in which one to all six acidic residues were systematically neutralized, often in various spatial configurations. There was no obvious correlation between the midpoint potentials for the oxidized/semiquinone couple and general electrostatic environment, although some differences were noted. However, the midpoint potential for the semiquinone/hydroquinone couple for each of the mutants was less negative than that of the wild type. These increases are strongly correlated with the number of acid to amide substitutions, with an average contribution of about 15 mV per substitution. Collectively, the unfavorable electrostatic environment provided by these acidic residues accounts for approximately one-third of the large midpoint potential shift for the semiquinone/hydroquinone couple that typifies the flavodoxin family, apparently through the destabilization of the flavin hydroquinone anion.

Amino Acids, Dicarboxylic↗
PubMed1995

Probing the role of electrostatic forces in the interaction of Clostridium pasteurianum ferredoxin with its redox partners.

The ability of several low-potential redox proteins to mediate electron transfer between Clostridium pasteurianum pyruvate-ferredoxin oxidoreductase and hydrogenase has been evaluated in a coupled enzymatic assay. The active electron mediators, whatever their structure, must have a reduction potential compatible with the two enzymes, but for proteins of similar potentials, a marked specificity is displayed by 2[4Fe-4S] ferredoxins of the clostridial type. Such ferredoxins are small proteins exchanging electrons with many enzymes involved in the metabolism of anaerobic bacteria. The forces underlying the interactions of ferredoxin with hydrogenase and pyruvate-ferredoxin oxidoreductase have been examined with an emphasis on electrostatics: site-directed mutagenesis experiments have been used to individually convert all conserved glutamates and aspartates of C. pasteurianum ferredoxin into either neutral or positively charged amino acids. Also, up to four of these residues have been replaced simultaneously. The biological activities of the resulting variants depend very little on the number and the distribution of the anionic side chains on the surface of the ferredoxin. Only those molecular forms for which the immediate environment of the clusters is perturbed, independently of the charge distribution, display variations in their catalytic properties. It is concluded that electron transfer between C. pasteurianum 2[4Fe-4S] ferredoxin and its partners is far less dependent on electrostatic interactions than in many other well-documented electron transfer systems.

Amino Acid Sequence↗
PubMed1993

Comparison of electrostatic interactions and of protein-protein orientations in electron-transfer reactions of plastocyanin with the triplet state of zinc cytochrome c and with zinc cytochrome c cation radical.

Photoinduced reduction of cupriplastocyanin by the triplet state of zinc cytochrome c (the "forward" reaction) and the subsequent thermal oxidation of cuproplastocyanin by zinc cytochrome c cation radical (the "back" reaction) at ionic strengths from 40 mM to 3.00 M are studied by laser kinetic spectroscopy (so-called flash photolysis). Variation of the bimolecular rate constants over the entire range of ionic strength cannot be explained in terms of monopole-monopole interactions between the protein molecules, but it can be explained in terms of monopole-monopole, monopole-dipole, and dipole-dipole interactions. Analysis of the kinetic results in terms of these electrostatic interactions reveals the overall protein-protein orientation for electron transfer. In both the forward and back reactions the exposed heme edge in zinc cytochrome c apparently abuts the negatively-charged (acidic) patch on the plastocyanin surface, which is remote from the copper atom, and not the electroneutral (hydrophobic) patch, which is proximate to the copper atom. The acidic patch is large, and this analysis cannot rule out a relatively small difference in protein-protein orientations for the forward and back reactions. These two reactions are compared with the previously studied reduction of cupriplastocyanin by ferrocytochrome c. Although native cytochrome c and its zinc derivative have very similar structural and electrostatic properties, the reactive forms of the cytochrome c/plastocyanin and zinc cytochrome c/plastocyanin complexes may adopt somewhat different protein-protein orientations or may adopt similar orientations but differ in dynamic properties.

Cations↗
PubMed1993

Electrostatic and nonpolar peptide-membrane interactions. Lipid binding and functional properties of somatostatin analogues of charge z = +1 to z = +3.

The interaction of four structurally related somatostatin analogues (effective electric charge +0.4 < or = < or = +3) with lipid membranes was studied with titration calorimetry and was compared with the functional activity of the peptides. Surface activity measurements provided average cross-sections of 70 or 135 A2, indicating that the cyclic molecules orient at the air-water interface with their ring system either parallel (z = +3) or perpendicular (z = +1) to the surface or switching between the two orientations according to the surface density (z = +2). The nonspecific binding of the peptides to sonified lipid vesicles was enthalpy-driven with a delta H of -4 to -7.5 kcal/mol. A consistent quantitative analysis of the binding isotherms was achieved by combining electrostatic attractions, calculated via the Gouy-Chapman theory, with a nonspecific surface partition equilibrium for the nonpolar interactions. The electrostatic attraction of the cationic peptides varied strongly according to the peptide charge. Due to the flat ring structure of the cyclic peptides, their true physical charge was sensed at the membrane surface, and no "charge screening" was observed. Peptide binding to the negative charged membrane was accompanied by a proton-uptake of the N-terminal amino group of 0.23-0.38 H+/peptide. Deviations from the theoretical prediction of 0.39 H+/peptide can be explained by a preferential binding of the nonprotonated species. The nonpolar interactions, as described by the surface partition coefficients of the four peptides, fell into a narrow range of K congruent to 50-230 M-1 whereas the apparent overall binding constants were between 200 and 5000 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

Role of electrostatic interactions in SH2 domain recognition: salt-dependence of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the Syk kinase and the single SH2 domain of the Src kinase.

SH2 domains are small protein domains that bind specifically to tyrosyl-phosphorylated sequences. Because phosphorylation contributes a large part of the binding free energy, it has been postulated that electrostatic interactions may play an important role in SH2 domain recognition. To test this hypothesis, we have examined the salt dependence of the interaction between tyrosyl-phosphorylated peptides and SH2 domains. The dependence of the binding constant, K(obs), on [NaCl] was shown to be strong for binding of the tandem SH2 domain of the Syk kinase (Syk-tSH2) to doubly phosphorylated peptides derived from immune-receptor tyrosine activation motifs (dpITAMs): the slopes of plots of log(K(obs)) versus log [NaCl], designated SK(obs), ranged from -2.6 +/- 0.1 to -3.1 +/- 0.2. Binding of the single SH2 domain of the Src kinase to its consensus singly phosphorylated peptide (sequence pYEEI where pY indicates a phosphotyrosine) was also highly dependent on [NaCl] with a SK(obs) value of -2.4 +/- 0.1. The ability of salt to disrupt the interactions between Syk-tSH2 and dpITAM peptides was shown to be anion-dependent with the inhibitory effect following the order: phosphate > Cl(-) > F(-). For the Syk-tSH2 system, interactions in the pY-binding pockets were shown to be responsible for a large portion of the total salt dependence: removal of either phosphate from the dpITAM peptide reduced the magnitude of SK(obs) by 40-60% and weakened binding by 2-3 orders of magnitude. Consistent with this finding, binding of the single amino acid Ac-pY-NH(2) was characterized by a large salt dependence of binding and was also dependent on the identity of the perturbing anion. The role of peptide residues C-terminal to the pY, which are implicated in determining the specificity of the phosphopeptide-SH2 domain interaction, was next probed by comparing the binding of the Src SH2 domain to a peptide containing the pYEEI sequence with that of a lower affinity variant pYAAI peptide: the magnitude of SK(obs) for the variant peptide was reduced to -1.3 +/- 0.1 as compared to -2.4 +/- 0.1 for the pYEEI peptide, indicating that in addition to pY, residues conferring peptide binding specificity contribute significantly to the salt dependence of SH2 domain binding. This study shows that electrostatic interactions play important roles not only in mediating pY recognition and binding but also in contributing to the specificity of the interactions between tyrosyl phosphopeptides and SH2 domains.

Amino Acid Sequence↗
PubMed1993

Correlation of the molecular electrostatic potential surface of an enzymatic transition state with novel transition-state inhibitors.

The transition state stabilized by nucleoside hydrolase from Crithidia fasciculata is characterized by nearly complete glycosidic bond cleavage and oxycarbonium character in the ribosyl group [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. The electrostatic potential surface of the transition state provides detailed information which should be useful in the design of transition-state analogues [Horestein, B. A., & Schramm, V. L. (1993) Biochemistry 32, 7089-7097]. The electrostatic potential surface of inosine at the transition state contains a distributed positive charge resulting from the oxycarbonium ion character of the ribosyl ring. The ribosyl ring pucker is 3'-exo as a result of the near sp2 hybridization at Cl' of the ribose ring. A series of transition-state analogues have been synthesized which incorporate single or combined features of the transition state. Each feature of the transition state was analyzed for its contribution to binding energy. Kinetic inhibition constants correlate with the similarity of the inhibitor to the experimentally determined transition-state structure. Dissociation constants for the substrate and products of the reaction of inosine, hypoxanthine, and ribose are 380, 6200, and 700 microM, respectively. A transition-state analogue was synthesized which contains the required hydroxyl groups of the ribose ring, the positive charge feature of the oxycarbonium ion, and a hydrophobic mimic of the purine ring. The inhibitor 1(S)-phenyl-1,4-dideoxy-1,4-iminoribitol acts as a competitive inhibitor with respect to inosine with a dissociation constant of 0.17 microM. In addition, the inhibitor exhibits slow-onset inhibition which provides a final equilibrium dissociation constant of approximately 0.03 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Role of electrostatic interactions in the binding of fluorescein by anti-fluorescein antibody 4-4-20.

Anti-fluorescein antibodies are excellent model systems for studying the biochemical basis of molecular recognition because a prodigious amount of both physico-chemical and structural information is available for these antibodies. Furthermore, recombinant single-chain antibodies have been produced for several anti-fluorescein antibodies, and site-specific mutagenesis studies have defined the energetic contributions of a number of key active-site residues. In previous studies, we determined the three-dimensional structure of an antigen-binding fragment of a high-affinity anti-fluorescein antibody (4-4-20) in complex with fluorescein. These studies showed that fluorescein binds tightly in an aromatic slot and participates in a network of electrostatic interactions. In this report, we examine the role of electrostatic interactions in the 4-4-20 antigen-combining site by observing the effects of pH on the fluorescence of fluorescein and antigen-binding affinity. These studies showed that the salt link between fluorescein and Arg-L34 in 4-4-20 probably accounts for about -1.5 kcal/mol-1 of the observed free energy of interaction. Furthermore, at pH 10 and higher, the affinity decreases by more than 100-fold (delta delta G degrees approximately equal to 3 kcal mol-1). We attributed this decrease to the ionization of Tyr-L32, which probably disrupts a hydrogen bond between tyrosine's hydroxyl group and fluorescein's phenylcarboxylate group. The fluorescence lifetime of the 4-4-20/fluorescein complex was determined at both pH 8 and pH 10.6. Only one lifetime component (0.38 ns) was observed at pH 8, while two components (0.3 and 3.4 ns) were observed at pH 10.6.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state.

The pH dependence of stability of staphylococcal nuclease was studied with two independent equilibrium thermodynamic approaches. First, by measurement of stability in the pH range 9 to 3.5 by fluorescence-monitored denaturation with urea (Delta), GdnHCl (Delta), and heat (Delta). Second, by numerical integration of H(+) titration curves (Delta) measured potentiometrically under native (100 mM KCl) and unfolding (6.0 M GdnHCl) conditions. The pH dependence of stability described by Delta, Delta, and Delta was comparable but significantly different from the one described by Delta. The decrease in Delta between pH 9 and pH 4 was 4 kcal/mol greater than the decrease in Delta, Delta, and Delta in the same pH range. In 6 M GdnHCl, all the ionizable groups titrated with the pK(a) values of model compounds. Therefore, Delta represents the free energy difference between the native state (N) and an ensemble of unstructured, or expanded, and highly screened conformations. In contrast, the shallower pH dependence of stability described by Delta and by Delta between pH 9 and 5 was consistent with the titration of histidines with depressed, nativelike pK(a) values in the denatured state (D). These depressed pK(a) values likely reflect long-range electrostatic interactions with the other 29 basic groups and are a consequence of the compact character of the D state. The steep change in Delta and Delta at pH < 5 suggests that near pH 5 the structural and thermodynamic character of the D state shifts toward a state in which acidic residues titrate with normal pK(a) values, presumably because the electrostatic interactions with basic residues are lost, maybe as a consequence of an expansion.

Acids↗
PubMed1992

Electrostatic changes at the actomyosin-subfragment 1 interface during force-generating reactions.

The ionic strength dependence of the binding of rabbit skeletal muscle myosin subfragment 1, S1, to F-actin in the presence of saturating concentrations of MgATP or MgADP was analyzed in order to determine the association constants at zero ionic strength [K(0)] and the products of the net effective electric charges (magnitude of zMzA) at the binding interfaces. K(0) and magnitude of zM A were 1 x 10(6) M-1 and 17 esu2 for S1-MgADP,P, and 5 x 10(7) M-1 and 7 esu2 for S1-MgADP, respectively, for binding to F-actin at 25 degrees C. At ionic strengths near physiological, the increase in affinity is close to 10(4)-fold for this transition that may correspond to force generation in muscle fibers. The large, from 17 to 7 esu2, decrease in the electrostatic contribution to binding appears to be correlated with a much larger increase in nonelectrostatic interactions, unlike the simpler transition of actin-bound S1-MgADP to S1, which appears to be due entirely to electrostatic changes [Highsmith, S. (1990) Biochemistry 29, 10690-10694]. These results for acto-S1-MgADP,P to acto-S1-MgADP suggest that a substantial transformation of the actin binding site on S1 occurs even if there is a translocation to a new interface.

Actins↗
PubMed1992

Relationship between electrostatics and redox function in human thioredoxin: characterization of pH titration shifts using two-dimensional homo- and heteronuclear NMR.

The electrostatic behavior of potentially titrating groups in reduced human thioredoxin was investigated using two-dimensional (2D) 1H and 15N nuclear magnetic resonance (NMR) spectroscopy. A total of 241 chemical shift titration curves were measured over the pH range of 2.1-10.6 from homonuclear 1H-1H Hartmann-Hahn (HOHAHA) and heteronuclear 1H-15N Overbodenhausen correlation spectra. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the determination of pKas for certain isolated ionizable groups, including the single histidine residue at position 43 (pKa = 5.5 +/- 0.1) and a number of aspartic and glutamic acid carboxylate groups. Many of the titration curves demonstrate complex behavior due to the effects of interacting titrating groups, the long range of electrostatic interactions through the protein interior, and, perhaps, pH-induced conformational changes on the chemical shifts. Unambiguous assignment of the pKas for most of the 38 potentially ionizing groups of human thioredoxin could therefore not be made. In addition, there was no clear evidence that Asp-26 titrates in a manner corresponding to that observed in the Escherichia coli protein [Dyson, H. J., Tennant, L. L., & Holmgren, A. (1991) Biochemistry 30, 4262-4268]. The pKas of the active site cysteines were measured, however, with Cys-32 having an anomalously low value of 6.3 +/- 0.1 and that of Cys-35 between 7.5 and 8.6. These pKas are in agreement with proposed mechanisms for redox catalysis of thioredoxin and previously measured pKas within the active site of E. coli thioredoxin [Kallis, G. B., & Holmgren, A. (1980) J. Biol. Chem. 255, 10261-10265]. The stabilization of a thiolate anion at physiological pH can be explained by the interaction of the S gamma of Cys-32 with the amide of Cys-35 observed in the previously determined high-resolution solution structure of reduced human thioredoxin [Forman-Kay, J. D., Clore, G. M., Wingfield, P. T., & Gronenborn, A. M. (1991) Biochemistry 30, 2685-2698].

Binding Sites↗
PubMed1994

Extent and rate of proton release by photosynthetic water oxidation in thylakoids: electrostatic relaxation versus chemical production.

The detailed chemical mechanism of the four steps of photosynthetic oxidation of two molecules of water to yield molecular oxygen plus four protons is under contention. The observed release of protons is a composite of the chemical production and more indirect reactions such as electrostatically induced shifts of acid/base equilibria of peripheral amino acids. In thylakoids we studied the extent and the rate (at microsecond time resolution) of proton release and uptake by each of the four oxidation steps. The pattern of net proton release in thylakoids varied drastically (between 0.3 and 2 H+/e-) as a function of pH. It differed substantially from the pH-dependent patterns of PSII-enriched membrane fragments and core particles, but the stepped progression toward release of dioxygen (the Kok parameter triple) was about the same. This implied an electrostatic origin of this variation and, within the observed limits, a lack of (inhibitory) feedback of the uncompensated charge on the electron transfer from the catalytic Mn cluster to TyrZ+. The rate of rapid proton transfer to the amphiphilic, surface-adsorbed indicator neutral red was proportional to its concentration. The shortest half-transfer time was 12 microseconds, substantially shorter than the time for electron transfer from Mn to TyrZ+ at any oxidation step. Rapid deprotonation thus occurred at the level of TyrZ+. By rapid deprotonation acts the four light-driven oxidation steps S0-->S1-->S2-->S3-->S4 created between 3.4 (at pH 7.4) and 4.5 (pH 6.3) bases per photosystem II.(ABSTRACT TRUNCATED AT 250 WORDS)

Electricity↗
PubMed1994

Crystal structure of the K12M/G15A triosephosphate isomerase double mutant and electrostatic analysis of the active site.

The crystal structure of the yeast triosephosphate isomerase (TIM) double mutant K12M/G15A has been solved to 2 A by X-ray diffraction, and the effects of changing the positively charged lysine to the neutral methionine have been analyzed. The mutant enzyme was crystallized in the presence of the tight-binding inhibitor phosphoglycolohydroxamate, under standard conditions for obtaining crystals of the enzyme-inhibitor complex. The crystals obtained were of the same crystal form as the unliganded wild-type enzyme. The three-dimensional structure confirms that the Lys-12 to Met mutation prevents the enzyme from binding substrate and reveals that the reason is electrostatic and not steric. The substrate-binding loop is in its open position and the Met side chain points away from the active site. Overall, the mutant structure is very similar to that of the wild-type unliganded enzyme. The electrostatic potential at the active site of the mutant enzyme is, however, very different from that of the wild type. It has been postulated previously that Lys-12 may play a role in stabilizing the negative charge in the transition state. This K12M/G15A structure suggests that the active-site Lys, which is strictly conserved, is required for TIM to be able to bind its dianionic substrate.

Amino Acid Sequence↗
PubMed1994

Proton linkage in formation of the cytochrome c-cytochrome c peroxidase complex: electrostatic properties of the high- and low-affinity cytochrome binding sites on the peroxidase.

The electrostatic character of cytochrome c-cytochrome c peroxidase complex formation has been studied by potentiometric titration between pH 5.5 and 7.75. Potentiometric data obtained at ionic strength > or = 100 mM were adequately analyzed in terms of 1:1 complex formation while the simplest model capable of fitting similar data obtained at lower ionic strength involves the assumption of two inequivalent binding sites for the cytochrome on the peroxidase. The stability of cytochrome c binding at the high-affinity site is ca. three orders of magnitude greater than that observed for the low-affinity site and is optimal between pH 6.75 and 7. The electrostatic properties of the two binding sites are distinctly different because, at most values of pH, binding of cytochrome c to the high-affinity site results in proton release while binding of the cytochrome to the low-affinity site results in proton uptake. Furthermore, binding of the cytochrome to the low-affinity site appears to be least stable in the pH range where binding to the high-affinity site is optimal. Interestingly, the binding parameters derived from these measurements were independent of temperature, consistent with a substantial entropic contribution to complex stability. Ferricytochrome c binds to the peroxidase with a slightly greater affinity than does ferrocytochrome c, and no evidence for specific anion effects on complex stability was observed. At low ionic strength (< or = 50 mM) and high pH (7.75), the interaction of the two proteins is more complex and cannot be adequately analyzed in terms of the two-site model.

Binding Sites↗