Synthesis and photophysical studies of covalently linked porphyrin-21-thiaporphyrin dyads

The synthesis and photophysical properties of four covalently linked unsymmetrical porphyrin dyads containing two different porphyrin cores such as N₄ and N₃S are reported. The covalently linked dyads were prepared by the coupling of appropriate porphyrin having ethynylphenyl functional group at meso-position with porphyrin having iodophenyl or bromo functional group at meso-position under mild palladium coupling conditions. The photophysical study indicated an intramolecular singlet-singlet energy transfer from N₄/ZnN₄ porphyrin sub-unit to N₃S porphyrin sub-unit in all four dyads with an efficiency of energy transfer process was typically ?97%. To probe the role of linker in through bond electronic communication between the two porphyrin sub-units in dyads, the linker was varied from diphenylethyne to phenylethyne and the study revealed that the energy transfer rates and efficiencies were much higher for phenylethyne-bridged porphyrin dyads compared with diphenylethyne-bridged porphyrin dyads.     

Do salt bridges stabilize proteins? A continuum electrostatic analysis

The electrostatic contribution to the free energy of folding was calculated for 21 salt bridges in 9 protein X-ray crystal structures using a continuum electrostatic approach with the DELPHI computer-program package. The majority (17) were found to be electrostatically destabilizing; the average free energy change, which is analogous to mutation of salt bridging side chains to hydrophobic isosteres, was calculated to be 3.5 kcal/mol. This is fundamentally different from stability measurements using pKa shifts, which effectively measure the strength of a salt bridge relative to 1 or more charged hydrogen bonds. The calculated effect was due to a large, unfavorable desolvation contribution that was not fully compensated by favorable interactions within the salt bridge and between salt-bridge partners and other polar and charged groups in the folded protein. Some of the salt bridges were studied in further detail to determine the effect of the choice of values for atomic radii, internal protein dielectric constant, and ionic strength used in the calculations. Increased ionic strength resulted in little or no change in calculated stability for 3 of 4 salt bridges over a range of 0.1-0.9 M. The results suggest that mutation of salt bridges, particularly those that are buried, to "hydrophobic bridges" (that pack at least as well as wild type) can result in proteins with increased stability. Due to the large penalty for burying uncompensated ionizable groups, salt bridges could help to limit the number of low free energy conformations of a molecule or complex and thus play a role in determining specificity (i.e., the uniqueness of a protein fold or protein-ligand binding geometry).     

Thermodynamic models, describing formation of "bridges" between nucleic acid molecules and liquid crystals

Three variants of the model for the formation of "bridges" between the nucleic acid molecules fixed in the structure of particles of liquid-crystalline dispersions were considered. What the three variants have in common is that the bridges represent polymeric chelate cross-links consisting of alternating molecules of daunomycin and copper ions. The differences between the three variants are that in the first variant, the bridges begin and end with daunomycin molecules that form a complex by the mechanism of external binding with nucleic acids; in the second variant, the bridges begin and end with copper ions coupled with the pairs of bases of nucleic acids; and in the third variant, the bridges begin with the daunomycin molecule and end with the copper ion. For each variant, a mathematical model was constructed, which describes the formation of bridges, and equations of binding were derived. The results of calculations were compared with the experimental data. Within the framework of each variant, the values of the energy of interaction between the daunomycin molecule and the copper ion in the bridge, the energy of interaction of the daunomycin molecule with the nucleic acid, and the length of the bridge were varied. For all variants, those values of the parameters were chosen that fit best the experimental data. The theoretical curves obtained using the three variants of the model agree rather well with the family of experimental curves. The best agreement between the theoretical and experimental data was obtained when the polymeric chelate bridge includes more than two daunomycin molecules.     

Use of intrinsic binding energy for catalysis by a cofactor-independent DNA enzyme

The concept of the Circe effect, according to which an enzyme's substrate-binding energy is utilized to destabilize the substrate towards the reaction transition state, has been shown to be a relevant catalytic strategy for naturally occurring protein enzymes and for two ribozymes that use nucleotide-based substrates and metal ion cofactors. We wished to investigate whether such a catalytic strategy extends even to divergent and unevolved catalysts constructed from biopolymers. We examined the properties of a small, in vitro selected, and cofactor-independent DNA enzyme, PS5.M, which catalyzes porphyrin metallation. The metallation reaction is unique, in that the energies for binding and for metallation of both the substrate and of a transition-state analogue (TSA) can be measured. We report that PS5.M, originally selected for binding to the TSA, displays the Circe effect in channeling a significant component of entropy-rich "intrinsic" binding energy to distort and to alter the basicity of the bound substrate. The study demonstrates that nucleic acids are, by themselves, capable of creating active sites for the catalysis of chemical reactions involving non-nucleotide substrates. Furthermore, the study of the metallation of the TSA provides a quantitative estimate of the effectiveness of such a compound in mimicking the true transition state for porphyrin metallation.     

Salt bridge as a gatekeeper against partial unfolding

Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native‐state partial unfolding in a cysteine‐free version of Escherichia coli ribonuclease H (RNase H*). Thermolysin cleaves a protruding loop of RNase H^* through transient partial unfolding under native conditions. Lys86 and Asp108 in RNase H^* form a partially buried salt bridge that tethers the protruding loop. Investigation of the global stability of K86Q/D108N RNase H^* showed that the salt bridge does not significantly contribute to the global stability. However, K86Q/D108N RNase H^* is greatly more susceptible to proteolysis by thermolysin than wild‐type RNase H^* is. The free energy for partial unfolding determined by native‐state proteolysis indicates that the salt bridge significantly increases the energy for partial unfolding by destabilizing the partially unfolded form. Double mutant cycles with single and double mutations of the salt bridge suggest that the partially unfolded form is destabilized due to a significant decrease in the interaction energy between Lys86 and Asp108 upon partial unfolding. This study demonstrates that, even in the case that a salt bridge does not contribute to the global stability, the salt bridge may function as a gatekeeper against partial unfolding that disturbs the optimal geometry of the salt bridge.     

The Preparation of Electrohydrodynamic Bridges from Polar Dielectric Liquids

Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and polar dielectric liquids. These bridges are unique from capillary bridges in that they exhibit extensibility beyond a few millimeters, have complex bi-directional mass transfer patterns, and emit non-Planck infrared radiation. A number of common solvents can form such bridges as well as low conductivity solutions and colloidal suspensions. The macroscopic behavior is governed by electrohydrodynamics and provides a means of studying fluid flow phenomena without the presence of rigid walls. Prior to the onset of a liquid bridge several important phenomena can be observed including advancing meniscus height (electrowetting), bulk fluid circulation (the Sumoto effect), and the ejection of charged droplets (electrospray). The interaction between surface, polarization, and displacement forces can be directly examined by varying applied voltage and bridge length. The electric field, assisted by gravity, stabilizes the liquid bridge against Rayleigh-Plateau instabilities. Construction of basic apparatus for both vertical and horizontal orientation along with operational examples, including thermographic images, for three liquids (e.g., water, DMSO, and glycerol) is presented.     

Rates of energy transfer between tryptophans and hemes in hemoglobin, assuming that the heme is a planar oscillator.

Using the Förster equations we have estimated the rate of energy transfer from tryptophans to hemes in hemoglobin. Assuming an isotropic distribution of the transition moments of the heme in the plane of the porphyrin, we computed the orientation factors and the consequent transfer rates from the crystallographic coordinates of human oxy- and deoxy-hemoglobin. It appears that the orientation factors do not play a limiting role in regulating the energy transfer and that the rates are controlled almost exclusively by the intrasubunit separations between tryptophans and hemes. In intact hemoglobin tetramers the intrasubunit separations are such as to reduce lifetimes to 5 and 15 ps/ns of tryptophan lifetime. Lifetimes of several hundred picoseconds would be allowed by the intersubunit separations, but intersubunits transfer becomes important only when one heme per tetramer is absent or does not accept transfer. If more than one heme per tetramer is absent lifetimes of more than 1 ns would appear.     

Photodynamics of excitation energy transfer in self-assembled dyads. Evidence for back transfer.

Three self-assembled photonic dyads comprising a zinc porphyrin donor and a free base acceptor have been studied by time-resolved fluorescence spectroscopy. The driving force of the assembly is the site selective binding of an imidazole connected to a free base porphyrin. Three spacers have been incorporated between the imidazole connector and the free base porphyrin, providing three different distances separating the donor and the acceptor. The high efficiencies and the rates of energy transfer in the set of dyads is consistent with the Forster energy transfer mechanism. Evidence for Forster back transfer has been obtained, and its efficiency and rate have been quantitatively evaluated for the first time.     

Regulation Mechanism of Excitation Energy Transfer in Phycobilisome-Thylakoid Membrane Complexes

Regulation mechanism of excitation energy transfer between phycobilisomes (PBS) and the photosynthetic reaction centres was studied by the state transition techniques in PBS-thylakoid membrane complexes. DCMU, betaine, and N-ethylmaleimide were applied to search for the details of energy transfer properties based on the steady fluorescence measurement and individual deconvolution spectra at state 2 or state 1. The closure of photosystem (PS) 2 did not influence on fluorescence yields of PS1, i.e., energy could not spill to PS1 from PS2. When the energy transfer pathway from PBS to PS1 was disturbed, the relative fluorescence yield of PS2 was almost the same as that of PS2 in complexes without treatment. If PBSs were fixed by betaine, the state transition process was restrained. Hence PBS may detach from PS2 and become associated to PS1 at state 2. Our results contradict the proposed "spill-over" or "PBS detachment" models and support the mobile "PBS model".     

Stable intercellular bridges in development: the cytoskeleton lining the tunnel

A wide variety of intercellular junctions that are involved with cell adhesion or signal transduction have been described in recent years. A widespread but less well-characterized type of intercellular junction is the stable intercellular bridge. Several organisms use stable intercellular bridges as cytoplasmic connections, probably to allow rapid transfer of information and organelles between cells. Here, the authors take a detailed look at the assembly of intercellular bridges called ring canals in the Drosophila germline and discuss how examination of mutants that disrupt Drosophila ovarian ring canal assembly indicates that these bridges are required for intercellular transport of cytoplasm.     

Studies on the interaction between tetraphenylporphyrin compounds and bovine serum albumin.

The interaction of three porphyrin compounds with bovine serum albumin (BSA) was examined by fluorescence emission spectra at the excitation wavelength 280 nm and in UV-Vis absorption spectra. Through fluorescence quenching experiments, it was confirmed that the combination of three porphyrin compounds with BSA was a single static quenching process. The binding constant K(A), the thermodynamic parameters enthalpy change (DeltaH(0)), Gibbs free energy change (DeltaG(0)) and entropy change (DeltaS(0)) were obtained. It was found that hydrophobic interaction played a main role in tetraphenylporphyrin (TPP) or tetraparacholophenylporphyrin (TClPP) binding to BSA, while tetraparamethoxyphenylporphyrin (TMEOPP) mainly based on van der Waals' force. According to F?ster energy transfer, the separate distance r, the energy transfer efficiency E and F?ster radium R(0) were calculated. The results obtained from the above experiments showed that three porphyrin compounds were tightly bound to BSA.     

Role of non-linear optics and multiple photon absorbance in enhancing sensitivity of enzyme-based chemical agent detectors

Real-time chemical sensors have been developed based on the binding of the analyte to monolayers of either porphyrin alone or porphyrins incorporated into the active site of enzymes. Binding of an analyte to porphyrin alone causes a redistribution of electrons in the porphyrin, altering the energy levels of the electrons which manifests as a change in the absorbance spectrum of the porphyrin. Porphyrins incorporated into the active site of enzymes such as cholinesterases are displaced when a competitive inhibitor such as nerve agents binds to the active site; this results in the porphyrin experiencing a different microenvironment than in the protein, resulting in a change in absorbance spectrum. Based on the Beer-Lambert relationship of concentration and absorbance, the limit of detection (LOD) for porphyrin-based sensors should be approximately 2 nM although LODs several orders of magnitude lower have been published. This increased sensitivity is explained as the result of multiple photon absorbance by the porphyrin and limiting self-quenching energy transfer reactions in the evanescent monolayer.     

A newly introduced salt bridge cluster improves structural and biophysical properties of de novo TIM barrels

Protein stability can be fine‐tuned by modifying different structural features such as hydrogen‐bond networks, salt bridges, hydrophobic cores, or disulfide bridges. Among these, stabilization by salt bridges is a major challenge in protein design and engineering since their stabilizing effects show a high dependence on the structural environment in the protein, and therefore are difficult to predict and model. In this work, we explore the effects on structure and stability of an introduced salt bridge cluster in the context of three different de novo TIM barrels. The salt bridge variants exhibit similar thermostability in comparison with their parental designs but important differences in the conformational stability at 25°C can be observed such as a highly stabilizing effect for two of the proteins but a destabilizing effect to the third. Analysis of the formed geometries of the salt bridge cluster in the crystal structures show either highly ordered salt bridge clusters or only single salt bridges. Rosetta modeling of the salt bridge clusters results in a good prediction of the tendency on stability changes but not the geometries observed in the three‐dimensional structures. The results show that despite the similarities in protein fold, the salt bridge clusters differently influence the structural and stability properties of the de novo TIM barrel variants depending on the structural background where they are introduced.     

Salt bridge stability in monomeric proteins.

Here, we present the results of continuum electrostatic calculations on a dataset of 222 non-equivalent salt bridges derived from 36 non-homologous high-resolution monomeric protein crystal structures. Most of the salt bridges in our dataset are stabilizing, regardless of whether they are buried or exposed, isolated or networked, hydrogen bonded or non-hydrogen bonded. One-third of the salt bridges in our dataset are buried in the protein core, with the remainder exposed to the solvent. The difference in the dielectric properties of water versus the hydrophobic protein interior cost buried salt bridges large desolvation penalties. However, the electrostatic interactions both between the salt-bridging side-chains, and between the salt bridges and charges in their protein surroundings, are also stronger in the interior, due to the absence of solvent screening. Even large desolvation penalties for burying salt bridges are frequently more than compensated for, primarily by the electrostatic interactions between the salt-bridging side-chains. In networked salt bridges both types of electrostatic interactions, those between the salt-bridging side-chains, and those between the salt bridge and its protein environment, are of similar magnitudes. In particular, a major finding of this work is that salt bridge geometry is a critical factor in determining salt bridge stability. Salt bridges with favorable geometrical positioning of the interacting side-chain charged groups are likely to be stabilizing anywhere in the protein structure. We further find that most of the salt bridges are formed between residues that are relatively near each other in the sequence.     

Reaction profile of the last step in cytochrome P-450 catalysis revealed by studies of model complexes

 A series of oxoiron(IV) porphyrin cation radical complexes was investigated as compound I analogs of cytochrome P-450. Both the spectroscopic features and the reactivities of the complexes in oxygen atom transfer to olefins were examined as a function of only one variable, the axial ligand trans to the oxoiron(IV) bond. The results disclosed two important kinetic steps – electron transfer from olefin to oxoiron(IV) and intramolecular electron transfer from metal to porphyrin radical – which are affected differently by the axial ligands. The large kinetic barrier of the latter step in the reaction of olefins with the perchlorato-bound oxoiron(IV) porphyrin cation radical complex enabled the trapping of a reaction intermediate in which the metal, but not the porphyrin radical, is reduced. The first electron transfer step is probably followed by σ-bond formation, which readily accounts for formation of isomerized organic products at low temperatures. It is finally postulated that part of the enhanced oxygenation activities of cytochrome P-450 monooxygenases and chloroperoxidases is due to a lowering of the energy barrier for the second electron transfer step via participation of their redox-active cysteinate ligand. Received: 16 January 1997 / Accepted: 24 May 1997     

Molecular dynamics simulation reveals a surface salt bridge forming a kinetic trap in unfolding of truncated Staphylococcal nuclease

Surface salt bridges are ubiquitous in globular proteins. Their contribution to protein stability has been extensively debated in the past decade. Here, molecular dynamics simulations are performed starting from a non-equilibrium state of Staphylococcal nuclease (SNase) with C-terminal truncation (SNaseDelta). The results indicate a key role in the unfolding of the surface salt bridge between arginine 105 and glutamate 135. Experimentally, SNaseDelta is known to be partially unfolded. However, in simulations over 1 ns at 300 K and over 500 ps at 400 K, SNaseDelta remains stable in the native-like folded conformation, the salt bridge hindering unfolding. When the potential function is altered so as to selectively weaken the salt bridge, which then breaks rapidly at 430 K, the protein starts to unfold. The results suggest that breaking of this salt bridge presents a significant barrier to the unfolding transition of SNaseDelta from a native-like state to the unfolded state. Potential of mean force calculations indicate that the barrier height for this transition is approximately 7 kcal/mol.     

Intercellular interactions preceding muscle cell fusion

Membrane interactions of myogenic cells of the 7-8 old chick embryos were investigated by electron microscopy. Tannic acid was used as specific fixative for biological membranes. Three types of specialized contacts can be revealed in fused myogenic cells: the first is like gap junctions, the second represents a pentalamellar structure, and the third is a so far non-described type of contact showing a membrane complex consisting of two parallel membranes arranged at a distance of approximately 15 nm and connected by electron-dense "bridges". The third type contact is revealed only by tannin-fixation. It is suggested that the "bridge" contact precedes the pentalamellar structure. A transition from the first type of contact to the second one is possible. The pentalamellar structure can be considered as an initial phase of fusion.     

Oxidizing intermediates in cytochrome P450 model reactions

Oxoiron(IV) porphyrin -cation radicals have been considered as the sole reactive species in the catalytic oxidation of organic substrates by cytochromes P450 and their iron porphyrin models over the past two decades. Recent studies from several laboratories, however, have provided experimental evidence that multiple oxidizing species are involved in the oxygen transfer reactions and that the mechanism of oxygen transfer is much more complex than initially believed. In this Commentary, reactive intermediates that have been shown or proposed to be involved in iron porphyrin complex-catalyzed oxidation reactions are reviewed. Particularly, the current controversy on the oxoiron(IV) porphyrin -cation radical as a sole reactive species versus the involvement of multiple oxidizing species in oxygen transfer reactions is discussed.Abbreviations F₅PhIO pentafluoroiodosylbenzene - m-CPBA m-chloroperbenzoic acid - OEP dianion of octaethylporphyrin - PhIO iodosylbenzene - PPAA peroxyphenylacetic acid - TDCPP dianion of meso-tetrakis(2,6-dichlorophenyl)porphyrin - TMP dianion of meso-tetramesitylporphyrin - TPFPP dianion of meso-tetrakis(pentafluorophenyl)porphyrin - TPP dianion of meso-tetraphenylporphyrin - TTPPP dianion of meso-tetrakis(2,4,6-triphenylphenyl)porphyrin     

The occurrence of intercellular bridges during oogenesis in the mouse

A fine structural analysis of fetal mouse ovaries reveals the presence of intercellular bridges between developing oocytes. These bridges, which connect two or more oocytes, are most frequently seen prior to the dictyate stage of meiotic prophase. The intercellular connections are limited by a tri-laminar membrane which is continuous with the oocyte plasmalemma. A characteristic feature of all bridges is the presence of an electron-dense material on the cytoplasmic side of the limiting membrane. Since this dense material is a constant and conspicuous component of the entire bridge, identification of these connections is possible in all planes of section. In cross section, the bridges are usually cylindrical, while in longitudinal section, a variety of configurations are observed. Oocytes connected by intercellular bridges exhibit a highly developed Golgi complex which is frequently localized in the region of the cytoplasmic continuities. Vesicular elements, apparently derived from the Golgi, are routinely observed within the boundaries of the bridges. Other cytoplasmic organelles, including rough and smooth endoplasmic reticulum, free ribosomes and mitochondria, are also seen in these bridges. The presence of these vesicles and organelles within intercellular bridges suggests that these connections may provide a means for transfer of organelles and other substances from one oocyte to another. It may be, therefore, that intercellular bridges are important for the nourishment and maturation of certain selected oocytes as well as for the synchronization of meiotic events.     

Protein stabilization by salt bridges: concepts, experimental approaches and clarification of some misunderstandings

Salt bridges in proteins are bonds between oppositely charged residues that are sufficiently close to each other to experience electrostatic attraction. They contribute to protein structure and to the specificity of interaction of proteins with other biomolecules, but in doing so they need not necessarily increase a protein's free energy of unfolding. The net electrostatic free energy of a salt bridge can be partitioned into three components: charge-charge interactions, interactions of charges with permanent dipoles, and desolvation of charges. Energetically favorable Coulombic charge-charge interaction is opposed by often unfavorable desolvation of interacting charges. As a consequence, salt bridges may destabilize the structure of the folded protein. There are two ways to estimate the free energy contribution of salt bridges by experiment: the pK(a) approach and the mutation approach. In the pK(a) approach, the contribution of charges to the free energy of unfolding of a protein is obtained from the change of pK(a) of ionizable groups caused by altered electrostatic interactions upon folding of the protein. The pK(a) approach provides the relative free energy gained or lost when ionizable groups are being charged. In the mutation approach, the coupling free energy between interacting charges is obtained from a double mutant cycle. The coupling free energy is an indirect and approximate measure of the free energy of charge-charge interaction. Neither the pK(a) approach nor the mutation approach can provide the net free energy of a salt bridge. Currently, this is obtained only by computational methods which, however, are often prone to large uncertainties due to simplifying assumptions and insufficient structural information on which calculations are based. This state of affairs makes the precise thermodynamic quantification of salt bridge energies very difficult. This review is focused on concepts and on the assessment of experimental methods and does not cover the vast literature.