Change In Protonation State Research Articles

PH-Dependent Phase Behavior of Carbohydrate-Based Gemini Surfactants. The Effects of Carbohydrate Stereochemistry, Head Group Hydrophilicity, and Nature of the Spacer

The pH-dependent phase behavior and hydroxide-ion adsorption ability of a series of (reduced) carbohydrate-based gemini surfactants were studied between pH 2 and 12. Static and dynamic light scattering were employed to address transitions in the aggregate morphologies and cryo-electron microscopy was used to provide further evidence for the morphologies present in solution. Changes in aggregate structure as a result of a change in solution pH and an accompanying change in protonation state or a change in molecular structure can be rationalized in terms of the variations in the packing parameter. In this paper we have focused our attention on the size of the carbohydrate moiety, the carbohydrate stereochemistry and the nature of the spacer (hydrophobic vs hydrophilic). At near neutral pH, most of the gemini surfactants form vesicles. Upon lowering of the pH, the vesicles undergo a transition toward wormlike micelles followed by a transition to spherical micelles. Upon increasing the solution pH, flocculation occurs due to charge neutralization followed at still higher pH by redispersion and charge reversal of the vesicles through the specific adsorption of hydroxide ions to the vesicular surface. Upon decreasing head group size at constant, but low, degrees of protonation, the packing parameter has a tendency to become larger than one resulting in the formation of inverted phases. Upon further decrease in the head group size, oil droplets are observed. In case of a hydrophobic spacer, the carbohydrate stereochemistry affects the pH of the transitions, but not the type of the transitions. By contrast, for a hydrophilic spacer, the pH of the transitions remains unaffected. Adsorption of hydroxide ions at basic pH follows similar trends, but was only found for vesicles and oil droplets. The large range of structural variations that we have examined allows a better understanding of the requirements for the phase transitions for carbohydrate-based gemini surfactants as well as for the physisorption of hydroxide ions to interfaces in general.

Redox-linked protonation state changes in cytochrome bc1 identified by Poisson–Boltzmann electrostatics calculations

Cytochrome bc 1 is a major component of biological energy conversion that exploits an energetically favourable redox reaction to generate a transmembrane proton gradient. Since the mechanistic details of the coupling of redox and protonation reactions in the active sites are largely unresolved, we have identified residues that undergo redox-linked protonation state changes. Structure-based Poisson–Boltzmann/Monte Carlo titration calculations have been performed for completely reduced and completely oxidised cytochrome bc 1. Different crystallographically observed conformations of Glu272 and surrounding residues of the cytochrome b subunit in cytochrome bc 1 from Saccharomyces cerevisiae have been considered in the calculations. Coenzyme Q (CoQ) has been modelled into the CoQ oxidation site (Q o-site). Our results indicate that both conformational and protonation state changes of Glu272 of cytochrome b may contribute to the postulated gating of CoQ oxidation. The Rieske iron–sulphur cluster could be shown to undergo redox-linked protonation state changes of its histidine ligands in the structural context of the CoQ-bound Q o-site. The proton acceptor role of the CoQ ligands in the CoQ reduction site (Q i-site) is supported by our results. A modified path for proton uptake towards the Q i-site features a cluster of conserved lysine residues in the cytochrome b (Lys228) and cytochrome c 1 subunits (Lys288, Lys289, Lys296). The cardiolipin molecule bound close to the Q i-site stabilises protons in this cluster of lysine residues.

Computational Analysis of the Membrane Association of Group IIA Secreted Phospholipases A2: A Differential Role for Electrostatics

Secreted phospholipases A2 (sPLA2's) are enzymes that hydrolyze glycerophospholipids at the sn-2 position, which leads to the production of lipid mediators of many cellular processes. These interfacial enzymes are regulated by their lipid specificity at two levels: membrane binding and substrate recognition. Different sPLA2's utilize different combinations of electrostatic and hydrophobic interactions to adsorb to membrane surfaces, which results in the wide range of membrane binding behaviors observed. Here, the finite difference Poisson Boltzmann (FDPB) method is used to quantitatively analyze the contribution of electrostatic interactions to the membrane association of two highly basic group II sPLA2's: Agkistrodon piscivorus piscivorus (AppD49) sPLA2 and nonpancreatic human group IIA (hGIIA) sPLA2. The calculations predict how membrane binding is affected by ionic strength, membrane composition, substitutions of residues in the enzymes, and the presence of calcium in the active site. In addition, the results provide molecular models for the membrane-associated forms of the enzymes. Furthermore, these models account for (1) changes in orientation and protonation state of both the native and charge reversal forms of the enzymes at the membrane surface and (2) the effect of protein/vesicle aggregation, as observed for hGIIA sPLA2. Importantly, the modeling quantitatively describes the complex membrane binding behaviors of these interfacial enzymes in terms of simple physical forces and provides structural information that is difficult to obtain experimentally. The computational analysis shows that nonspecific electrostatic interactions not only play a major role in recruiting these enzymes to membrane surfaces but also orient the enzymes for productive catalysis at the membrane interface.

The Triplex-Hairpin Transition in Cytosine-Rich DNA

We present a theoretical study of the self-complementary single-stranded 30-mer d (TC*TTC*C*TTTTCCTTCTC*CCGAGAAGGTTTT) (PDB ID: 1b4y) that was designed to form an intramolecular triplex by folding back twice on itself. At neutral pH the molecule exists in a duplex hairpin conformation, whereas at acidic pH the cytosines labeled by an asterisk (*) are protonated, forming Hoogsteen hydrogen bonds with guanine of a GC Watson-Crick basepair to generate a triplex. As a first step in an investigation of the energetics of the triplex-hairpin transition, we applied the Bashford-Karplus multiple site model of protonation to calculate the titration curves for the two conformations. Based on these data, a two-state model is used to study the equilibrium properties of transition. Although this model properly describes the thermodynamics of the protonation-deprotonation steps that drive the folding-unfolding of the oligomer, it cannot provide insight into the time-dependent mechanism of the process. A series of molecular dynamics simulations using the ff94 force field of the AMBER 6.0 package was therefore run to explore the dynamics of the folding/unfolding pathway. The molecular dynamics method was combined with Poisson-Boltzmann calculations to determine when a change in protonation state was warranted during a trajectory. This revealed a sequence of elementary protonation steps during the folding/unfolding transition and suggests a strong coupling between ionization and folding in cytosine-rich triple-helical triplexes.

Mid- to low-frequency Fourier transform infrared spectra of S-state cycle for photosynthetic water oxidation in Synechocystis sp. PCC 6803.

Flash-induced Fourier transform infrared (FTIR) difference spectra for the four-step S-state cycle and the effects of global (15)N- and (13)C-isotope labeling on the difference spectra were examined for the first time in the mid- to low-frequency (1200-800 cm(-1)) as well as the mid-frequency (1700-1200 cm(-1)) regions using photosystem (PS) II core particles from cyanobacterium Synechocystis sp. PCC 6803. The difference spectra clearly exhibited the characteristic vibrational features for each transition during the S-state cycling. It is likely that the bands that change their sign and intensity with the S-state advances reflect the changes of the amino acid residues and protein matrices that have functional and/or structural roles within the oxygen-evolving complex (OEC). Except for some minor differences, the trends of S-state dependence in the 1700-1200 cm(-1) frequency spectra of the PS II cores from Synechocystis were comparable to that of spinach, indicating that the structural changes of the polypeptide backbones and amino acid side chains that occur during the oxygen evolution are inherently identical between cyanobacteria and higher plants. Upon (13)C-labeling, most of the bands, including amide I and II modes and carboxylate stretching modes, showed downward shifts; in contrast, (15)N-labeling induced isotopic shifts that were predominantly observed in the amide II region. In the mid- to low-frequency region, several bands in the 1200-1140 cm(-1) region were attributable to the nitrogen- and/or carbon-containing group(s) that are closely related to the oxygen evolution process. Specifically, the putative histidine ligand exhibited a band at 1113 cm(-1) which was affected by both (15)N- and (13)C-labeling and showed distinct S-state dependency. The light-induced bands in the 900-800 cm(-1) region were downshifted only by (13)C-labeling, whereas the bands in the 1000-900 cm(-1) region were affected by both (15)N- and (13)C-labeling. Several modes in the mid- to low-frequency spectra were induced by the change in protonation state of the buffer molecules accompanied by S-state transitions. Our studies on the light-induced spectrum showed that contributions from the redox changes of Q(A) and the non-heme iron at the acceptor side and Y(D) were minimal. It was, therefore, suggested that the observed bands in the 1000-800 cm(-1) region include the modes of the amino acid side chains that are coupled to the oxidation of the Mn cluster. S-state-dependent changes were observed in some of the bands.

Acid−Base Equilibria in Rhodopsin: Dependence of the Protonation State of Glu134 on Its Environment

Glutamic acid E134 in rhodopsin is part of a highly conserved triad, D(E)RY, located near the cytoplasmic lipid/water interface in transmembrane helix 3 of G protein-coupled receptors (GPCRs). A large body of experimental evidence suggests that the protonation of E134 plays a role in the mechanism of activation of rhodopsin and other GPCRs as well. For E134 to change its protonation state, its pK(a) value must shift from values below physiological pH to higher values. Because of the proximity of the triad to the lipid/water interface, it was hypothesized that a change in solvent around E134 from water to lipid could induce such a shift in pK(a). To test this hypothesis, the pK(a) values of the titratable amino acid residues in rhodopsin have been calculated and the change in solvent around E134 was modeled by shifting the position of the lipid/water interface. The approach used to carry out the pK(a) calculations takes into account the partial immersion of transmembrane proteins in lipid. Qualitative experimental evidence is available for several residues regarding their likely protonation state in rhodopsin at or near physiological pH. Comparison of the calculated pK(a) values with these experimental findings shows good agreement between the two. Notably, glutamic acids E122 and E181 were found to be protonated. The pK(a) values were then calculated for a range of lipid/water interface positions. Although the surrounding solvent of several titratable residues changed from water to lipid in this range, leading to pK(a) shifts in most cases, only for E134 would the shift lead to a change in protonation state at physiological pH. Thus, our results show that the protonation state of E134 is particularly sensitive to its environment. This sensitivity together with the location of E134 near the actual position of the lipid/water interface could be a strategic element in the mechanism of activation of rhodopsin.

Observation of the Equilibrium CuB-CO Complex and Functional Implications of the Transient Hemea3 Propionates in Cytochromeba3-CO from Thermus thermophilus: FOURIER TRANSFORM INFRARED (FTIR) AND TIME-RESOLVED STEP-SCAN FTIR STUDIES

We report the first evidence for the existence of the equilibrium Cu(B)1+-CO species of CO-bound reduced cytochrome ba(3) from Thermus thermophilus at room temperature. The frequency of the C-O stretching mode of Cu(B)1+-CO is located at 2053 cm(-1) and remains unchanged in H(2)O/D(2)O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the Cu(B) histidine ligands. The data and conclusions reported here are in contrast to the changes in protonation state of Cu(B)-His-290, reported recently (Das, T. K., Tomson, F. K., Gennis, R. B., Gordon, M., and Rousseau, D. L. (2001) Biophys. J. 80, 2039-2045 and Das, T. P., Gomes, C. M., Teixeira, M., and Rousseau, D. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9591-9596). The time-resolved step-scan FTIR difference spectra indicate that the rate of decay of the transient Cu(B)1+-CO complex is 34.5 s(-1) and rebinding to heme a(3) occurs with k(2) = 28.6 s(-1). The rate of decay of the transient Cu(B)1+-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a(3) propionates (COOH). The nu(C-O) of the transient Cu(B)1+-CO species is the same as that of the equilibrium Cu(B)1+-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu(B) between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.

Vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans: FTIR and electrochemical studies on Tyr-D4-labeled and on Tyr280His and Tyr35Phe mutant enzymes.

A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer. Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines. Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes. The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1). A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine. Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra. Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276. The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine. The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding. The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center. Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected. In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed. Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B). Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV. Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase.

Calculation of Weak Protein-Protein Interactions: The pH Dependence of the Second Virial Coefficient

Interactions between proteins are often sufficiently weak that their study through the use of conventional structural techniques becomes problematic. Of the few techniques capable of providing experimental measures of weak protein-protein interactions, perhaps the most useful is the second virial coefficient, B 22, which quantifies a protein solution's deviations from ideal behavior. It has long been known that B 22 can in principle be computed, but only very recently has it been demonstrated that such calculations can be performed using protein models of true atomic detail ( Biophys. J. 1998, 75:2469–2477). The work reported here extends these previous efforts in an attempt to develop a transferable energetic model capable of reproducing the experimental trends obtained for two different proteins over a range of pH and ionic strengths. We describe protein-protein interaction energies by a combination of three separate terms: (i) an electrostatic interaction term based on the use of effective charges, (ii) a term describing the electrostatic desolvation that occurs when charged groups are buried by an approaching protein partner, and (iii) a solvent-accessible surface area term that is used to describe contributions from van der Waals and hydrophobic interactions. The magnitude of the third term is governed by an adjustable, empirical parameter, γ, that is altered to optimize agreement between calculated and experimental values of B 22. The model is applied separately to the proteins lysozyme and chymotrypsinogen, yielding optimal values of γ that are almost identical. There are, however, clear difficulties in reproducing B 22 values at the extremes of pH. Explicit calculation of the protonation states of ionizable amino acids in the 200 most energetically favorable protein-protein structures suggest that these difficulties are due to a neglect of the protonation state changes that can accompany complexation. Proper reproduction of the pH dependence of B 22 will, therefore, almost certainly require that account be taken of these protonation state changes. Despite this problem, the fact that almost identical γ values are obtained from two different proteins suggests that the basic energetic formulation used here, which can be evaluated very rapidly, might find use in dynamical simulations of weak protein-protein interactions at intermediate pH values.

Nature of cysteine-based Re(V)=O(N2S2) radiopharmaceuticals at physiological pH ascertained by investigation of a new complex with a meso N2S2 ligand having carboxyl groups anti to the oxo group.

X-ray structural characterization of a new isomer of ReO(TMECH3) revealed that it is anti-ReO(DL-TMECH3) (1). (DL-TMECH6 is meso-tetramethyl-ethylene-dicysteine prepared from racemic penicillamine (penH4), the subscript on H indicating the number of dissociable protons; anti denotes the geometric isomer having both carboxyl groups anti to the oxo ligand.) In 1, one carboxyl is deprotonated and coordinated trans to the oxo ligand, and the other is protonated and dangling. The 1H NMR spectrum (assigned by 2D methods) of 1 at pH 4 in aqueous solution revealed that the structure of 1 is the same as in the solid state except for deprotonation of the dangling carboxyl group, affording the monoanion. All chelate ring protons and methyl groups are inequivalent and give sharp signals. As the pH was raised above 7, the 1D 1H NMR signals of the monoanion broadened. Broadening was severe for the methyl and ethylene signals of the tridentate half of the monoanion, and these signals were replaced by new signals for the dianion. The changes suggested a rate process that was intermediate on the NMR time scale, such as CO2- ligation/deligation. With increasing pH the dianion signals sharpened up to pH approximately 8 and then broadened up to pH approximately 10. Finally, the spectrum at pH 10.8 showed only half the number of signals. Each signal was at the midpoint shift between two corresponding signals observed at lower pH, indicating a time averaging between the halves of the DL-TMEC ligand, but no change in protonation state. Two Re=O stretching bands (923 and 933 cm-1) with a constant intensity ratio of approximately 1 were observed for the dianion. These results can be explained if the dianion exists detectably only as a NH-deprotonated/carboxyl-deligated form having two conformers. The conformers differ in the N lone pair (NLp) orientation (either endo or exo with respect to the oxo ligand) and thus have slightly different Re=O stretching frequencies. Although they can be detected by resonance Raman spectroscopy, the conformers are indistinguishable by NMR spectroscopy because NLp inversion (and hence conformer interconversion) is very fast. Interchange of the NH and NLp sites affects the NMR spectra. At pH 8.3 the signals of the dianion are sharpest because interchange is slowest. Below and above pH 8.3, the signals are broader because acid and base catalysis, respectively, increase the rate of interchange between the NH and NLp sites.

A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease: A TRANSMEMBRANE POTENTIAL PERTURBS CARBOXYLIC ACID RESIDUES

In chemiosmotic coupling, a transmembrane ion gradient is used as the source of energy to drive reactions. This process occurs in all cells, but the microscopic mechanism is not understood. Here, Escherichia coli lactose permease was used in a novel spectroscopic method to investigate the mechanism of chemiosmotic coupling in secondary active transporters. To provide a light-triggered electrochemical gradient, bacteriorhodopsin was co-reconstituted with the permease, and reaction-induced Fourier transform-infrared spectra were obtained from the co-reconstituted samples. The bacteriorhodopsin contributions were subtracted from these data to give spectra reflecting permease conformational changes that are induced by an electrochemical gradient. Positive bands in the 1765-1730 cm(-1) region are attributable to carboxylic acid residues in the permease and are consistent with changes of pK(a), protonation state, or environment. This is the first direct information concerning gradient-induced structural changes in the permease at the single amino acid level. Ultimately, these structural changes facilitate galactoside binding and may be involved in the storage of free energy.

Protonation state and structural changes of the tetrapyrrole chromophore during the Pr --> Pfr phototransformation of phytochrome: a resonance Raman spectroscopic study.

The photoconversion of phytochrome (phytochrome A from Avena satina) from the inactive (Pr) to the physiologically active form (Pfr) was studied by near-infrared Fourier transform resonance Raman spectroscopy at cryogenic temperatures, which allow us to trap the intermediate states. Nondeuterated and deuterated buffer solutions were used to determine the effect of H/D exchange on the resonance Raman spectra. For the first time, reliable spectra of the "bleached" intermediates meta-R(A) and meta-R(C) were obtained. The vibrational bands in the region 1300-1700 cm(-)(1), which is particularly indicative of structural changes in tetrapyrroles, were assigned on the basis of recent calculations of the Raman spectra of the chromophore in C-phycocyanin and model compounds [Kneip, C., Hildebrandt, P., Németh, K., Mark, F., Schaffner, K. (1999) Chem. Phys. Lett. 311, 479-485]. The experimental resonance Raman spectra Pr are compatible with the Raman spectra calculated for the protonated ZZZasa configuration, which hence is suggested to be the chromophore structure in this parent state of phytochrome. Furthermore, marker bands could be identified that are of high diagnostic value for monitoring structural changes in individual parts of the chromophore. Specifically, it could be shown that not only in the parent states Pr and Pfr but also in all intermediates the chromophore is protonated at the pyrroleninic nitrogen. The spectral changes observed for lumi-R confirm the view that the photoreaction of Pr is a Z --> E isomerization of the CD methine bridge. The subsequent thermal decay reaction to meta-R(A) includes relaxations of the CD methine bridge double bond, whereas the formation of meta-R(C) is accompanied by structural adaptations of the pyrrole rings B and C in the protein pocket. The far-reaching similarities between the chromophores of meta-R(A) and Pfr suggest that in the step meta-R(A) --> Pfr the ultimate structural changes of the protein matrix occur.

Characterization of the photoreduction of the secondary quinone Q B in the photosynthetic reaction center from Rhodobacter capsulatus with FTIR spectroscopy

The photoreduction of the secondary quinone acceptor Q B in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter ( Rb.) capsulatus has been investigated by light-induced FTIR difference spectroscopy in 1H 2O and 2H 2O. The Q − B/Q B FTIR spectra reflect reorganization of the protein upon electron transfer, changes of protonation state of carboxylic acid groups, and (semi)quinone–protein interactions. As expected from the conservation of most of the amino acids near Q B in the RCs from Rb. capsulatus and Rb. sphaeroides, several protein and quinone IR bands are common to both spectra, e.g., the 1728 cm −1 band is assigned to proton uptake by a carboxylic acid residue, most probably Glu L212 as previously proposed for Rb. sphaeroides RCs. However, noticeable changes are observed at 1709 cm −1 (deprotonation of a Glu or Asp residue), 1674 and 1659 cm −1 (side chain and/or backbone), around 1540 cm −1 (amide II), and in the semiquinone absorption range. This FTIR study demonstrates that the environment of the secondary quinone in Rb. capsulatus is close but not identical to that in Rb. sphaeroides suggesting slight differences in the structural organization of side chains and/or ordered water molecules near Q B.

A Light-Activated Antibody Catalyst

A catalytic antibody for a multistep Norrish type II photochemical reaction was investigated. Absorption of light energy by α-ketoamide substrate 1b produced a high-energy biradical intermediate, that was then directed by the antibody microenvironment to form tetrahydropyrazine 13 with a kcat of 1.4 × 10-3 min-1 at 280 nm irradiation and an enantiomeric excess of 78%. Antibody-catalyzed reactions performed with radiolabeled substrate indicated that little self-inactivation (6.8 mol % covalent modification after four turnovers per antibody) occurred. The singular product obtained in the antibody-catalyzed reaction was not observed in the uncatalyzed reaction unless the pH was lowered below 4. Studies suggested that the interplay of conformational control and chemical catalysis were responsible for the high specificity. A change in protonation state of the antibody was correlated with the inclusion of a new reaction pathway in the antibody-catalyzed reaction, indicating that general-base catalysis was invol...

Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy.

Green Fluorescent Protein (GFP) is a bioluminescence protein from the jelly fish Aequorea victoria. It can exist in at least two spectroscopically distinct states: GFP395 and GFP480, with peak absorption at 395 and 480 nm, respectively, presumably resulting from a change in the protonation state of the phenolic ring of its chromophore. When GFP is formed upon heterologous expression in Escherichia coli, its chromophore is mainly present as the neutral species. UV and visible light convert (the chromophore of) GFP quantitatively from this neutral- into the anionic form. On the basis of X-ray diffraction, it was recently proposed (Brejc, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2306-2311; Palm, G. J. et al. (1997) Nat. Struct. Biol. 4, 361-365) that the carboxylic group of Glu222 functions as the proton acceptor of the chromophore of GFP, during the transition from the neutral form (i.e., GFP395) to the ionized form (GFP480). However, X-ray crystallography cannot detect protons directly. The results of FTIR difference spectroscopy, in contrast, are highly sensitive to changes in the protonation state between two conformations of a protein. Here we report the first characterization of GFP, and its photoconversion, with FTIR spectroscopy. Our results clearly show the change in protonation state of the chromophore upon photoconversion. However, they do not provide indications for a change of the protonation state of a glutamate side chain between the states GFP395 and GFP480, nor for an isomerization of the double bond that forms part of the link between the two rings of the chromophore.

The pH-Induced Release of Iron from Transferrin Investigated with a Continuum Electrostatic Model

A reduction in pH induces the release of iron from transferrin in a process that involves a conformational change in the protein from a closed to an open form. Experimental evidence suggests that there must be changes in the protonation states of certain, as yet not clearly identified, residues in the protein accompanying this conformational change. Such changes in protonation states of residues and the consequent changes in electrostatic interactions are assumed to play a large part in the mechanism of release of iron from transferrin. Using the x-ray crystal structures of human ferri- and apo-lactoferrin, we calculated the pK a values of the titratable residues in both the closed (iron-loaded) and open (iron-free) conformations with a continuum electrostatic model. With the knowledge of a residue’s pK a value, its most probable protonation state at any specified pH may be determined. The preliminary results presented here are in good agreement with the experimental observation that the binding of ferric iron and the synergistic anion bicarbonate/carbonate results in the release of approximately three H + ions. It is suggested that the release of these three H + ions may be accounted for, in most part, by the deprotonation of the bicarbonate and residues Tyr-92, Lys-243, Lys-282, and Lys-285 together with the protonation of residues Asp-217 and Lys-277.

Redox- and anion-linked protonation sites in horseradish peroxidase: analysis of distal haem pocket mutants.

We have investigated the effects of mutations at residues His-42, Arg-38 and Phe-41 in the distal haem pocket of horseradish peroxidase on the changes in protonation state that accompany redox- and ligand-linked changes to the haem group. The mutations H42L and R38L result in the loss of a characteristic pH dependency in the visible spectrum of the ferrous form and a diminished dependency of the midpoint redox potential of the haem group on pH. The results support the view that His-42, with its pK probably modulated by Arg-38, provides the protonation site on the reduced enzyme that is responsible for these pH dependencies. The mutations H42L and R38L also have major effects on the binding of cyanide to the haem. We have already reported that binding of cyanide to the ferrous forms of these mutants becomes too weak to be measurable [Meunier, Rodriguez-Lopez, Smith, Thorneley and Rich (1995) Biochemistry 34, 14687-14692]. The pH dependency of the rate constants for binding of cyanide to the oxidized form of H42L suggests that CN- is the kinetically active species, in contrast with wild-type horseradish peroxidase, where HCN is the binding form. For the R38L variant, the pH dependency of cyanide binding suggests that the pK of His-42 in the absence of Arg-38 is raised to 7.5-8, in the oxidized form. In contrast with these changes, the mutant F41A exhibits cyanide-binding behaviour that is similar to that of the wild type, both in its oxidized and reduced forms. However, the rate constant for carbon monoxide recombination increases substantially, suggesting that the access route for carbon monoxide, but not for cyanide, is perturbed by this amino acid substitution.

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon QB photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 → Asn replacement on QB−/QB FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 → Asp, Arg M233 → Cys, or Arg H177 → His suppressor mutation. The proton uptake by Glu L212 upon QB− formation, as reflected by the positive band at 1728 cm−1, is increased in the Asn M44 → Asp and Arg H177 → His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 → Asn mutant RCs. Only the Arg M233 → Cys suppressor mutation affected the 1728 cm−1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 → Asn lesion.

Incorporating protein conformational flexibility into the calculation of pH-dependent protein properties

A method for combining calculations of residue pKa's with changes in the position of polar hydrogens has been developed. The Boltzmann distributions of proton positions in hydroxyls and neutral titratable residues are found in the same Monte Carlo sampling procedure that determines the amino acid ionization states at each pH. Electrostatic, Lennard-Jones potentials, and torsion angle energies are considered at each proton position. Many acidic and basic residues are found to have significant electrostatic interactions with either a water- or hydroxyl-containing side chain. Protonation state changes are coupled to reorientation of the neighboring hydroxyl dipoles, resulting in smaller free energy differences between neutral and ionized residues than when the protein is held rigid. Multiconformation pH titration gives better agreement with the experimental pKa's for triclinic hen egg lysozyme than conventional rigid protein calculations. The hydroxyl motion significantly increases the protein dielectric response, making it sensitive to the composition of the local protein structure. More than one conformer per residue is often found at a given pH, providing information about the distribution of low-energy lysozyme structures.

Thermodynamics of an Intramolecular DNA Triple Helix: A Calorimetric and Spectroscopic Study of the pH and Salt Dependence of Thermally Induced Structural Transitions

We have characterized thermodynamically the melting transitions of a DNA 31-mer oligonucleotide (5′-GAAGAGGTTTTTCCTCTTCTTTTTCTTCTCC-3′) which is designed to fold into an intramolecular triple helix. The first 19 residues fold back on themselves to form an antiparallel Watson-Crick hairpin duplex with a T5loop. The 3′-terminal seven residues, which are connected to the Watson-Crick hairpin duplex by a second T5loop, form Hoogsteen interactions in the major groove of the Watson-Crick hairpin.From ultraviolet (UV) melting studies we find that the 31-mer exhibits either one or two transitions, depending on solution conditions. We use pH- and temperature-dependent circular dichroism (CD) to assign the initial and final states associated with each transition. We find that the disruption of the Hoogsteen hairpin is accompanied by a release of protons and an uptake of sodium ions while the disruption of the Watson-Crick hairpin is accompanied by a release of sodium ions with no change in protonation state. From these data, we construct a phase diagram for this intramolecular DNA triple helix as a function of pH, sodium ion concentration, and temperature. We characterize the energetics of each transition using a van't Hoff analysis and differential scanning calorimetry (DSC). Significantly, the DSC data provide a model-independent thermodynamic characterization of the thermally induced transitions of this triplex.By combining the spectroscopic and calorimetric data, we develop a semi-empirical model which describes the state of the 31-mer as a function of pH, sodium ion concentration, and temperature. With this model we successfully predict characteristics of the 31-mer, which are beyond the data which are used in establishing the model (for example, the salt dependence of the apparent pKaof the Hoogsteen strand). This semi-empirical model may serve as a prototype for developing a method to predict the phase diagrams of intramolecular triple helix systems.