On the dielectric spectra of proteins in conducting media

The effect of conductivity on the dielectric measurements of proteins is studied. For that purpose the dielectric spectra (0.03–13 MHz) of serum albumin and myoglobin in solutions of varying conductivities were recorded. The results presented confirm that Maxwell's prediction of a threshold frequency in conducting materials also holds for protein solutions. The threshold frequency of a serum albumin solution is experimentally determined and the ionic screening of the electric field when performing dielectric spectra of these samples discussed. Three distinct frequency regions must be considered: a low frequency region where the sample behaves like a conductor; an intermediate region centered around the threshold frequency where the free charges partially screen the fixed charges; and a high frequency region where the sample behaves like a good dielectric. Dielectric measurements in the low frequency region defined above, are not possible.     

Structure-independent cross-validation between residual dipolar couplings originating from internal and external orienting media

Lanthanide-substituted calcium binding proteins are known to partially orient in high magnetic fields. Orientation provides residual dipolar couplings (rdc's). Two of these systems, Tm³⁺- and Dy³⁺-substituted calbindin D_9k, dissolved in an external orienting medium (nonionic liquid crystalline phase) provide rdc values which are the sum of those induced by the lanthanides and by the liquid crystalline phase on the native calcium binding protein. This structure-independent check shows the innocence of the orienting medium with respect to the structure of the protein in solution. Furthermore, the simultaneous use of lanthanide substitution and external orienting media provides a further effective tool to control and tune the orientation tensor.     

De novo determination of internuclear vector orientations from residual dipolar couplings measured in three independent alignment media

The straightforward interpretation of solution state residual dipolar couplings (RDCs) in terms of internuclear vector orientations generally requires prior knowledge of the alignment tensor, which in turn is normally estimated using a structural model. We have developed a protocol which allows the requirement for prior structural knowledge to be dispensed with as long as RDC measurements can be made in three independent alignment media. This approach, called Rigid Structure from Dipolar Couplings (RSDC), allows vector orientations and alignment tensors to be determined de novo from just three independent sets of RDCs. It is shown that complications arising from the existence of multiple solutions can be overcome by careful consideration of alignment tensor magnitudes in addition to the agreement between measured and calculated RDCs. Extensive simulations as well applications to the proteins ubiquitin and Staphylococcal protein GB1 demonstrate that this method can provide robust determinations of alignment tensors and amide N-H bond orientations often with better than 10 degrees accuracy, even in the presence of modest levels of internal dynamics.     

Vitrification media: toxicity,permeability, and dielectric properties

The aim of this study was to select a cryoprotectant for use in attempts to preserve tissues and organs by vitrification. The first step was to select a cell line with which to compare the toxicity of a range of commonly used cryoprotectants. An immortal vascular endothelial cell (ECV304) was exposed to vitrifying concentrations of four cryoprotectants: dimethyl sulfoxide (Me(2)SO; 45% w/w); 2,3 butanediol (BD; 32%); 1,2-propanediol (PD; 45%); and ethanediol (ED; 45%). Three times of exposure (1, 3, and 9 min) and two temperatures (22 and 2-4 degrees C) were studied. After removal of the cryoprotectant, the ability of the cells to adhere and divide in culture over a 2-day period was measured and expressed as a Cell Survival Index (CSI). There was no measurable loss of cells after exposure to the four cryoprotectants but 3-min exposure to BD, PD, or Me(2)SO at room temperature completely destroyed the ability of the cells to adhere and divide in culture. In contrast, exposure to all four cryoprotectants at 2-4 degrees C for up to 9 min permitted the retention of significant cell function, the CSIs, as a proportion of control, being 76.3+/-7.0% for BD, 63.6+/-7.1% for PD, 37.0+/-4.1 for Me(2)SO, and 33.2+/-3.0 for ED. The permeability properties of the cells for these four cryoprotectants was also measured at each temperature. Permeability to water was high, L(p) approximately equal 10(-7) cm/s/atm at 2-4 degrees C with all the cryoprotectants, but there were substantial differences in solute permeability: BD and PD were the most permeable at 2-4 degrees C (P(s)=4.1 and 3.0 x 10(-6) cm/s, respectively). Equilibration of intracellular cryoprotectant concentration was rapid, due in part to high water permeability; the cells were approximately 80% of their physiological volume after 10 min. Treatment at 2-4 degrees C with BD was the least damaging, but PD was not significantly worse. Exposure to vitrifying concentrations of ED and Me(2)SO, even at 2-4 degrees C, was severely damaging. Segments of rabbit carotid artery were treated with vitrifying concentrations of each of the two most favorable cryoprotectants, BD and PD, for 9 min. It was shown that each cryoprotectant reduced smooth muscle maximum contractility to a similar extent and abolished the acetylcholine response. However, vital staining revealed that exposure to BD also caused substantial damage to the endothelial lining, whereas the endothelium was completely intact after PD exposure, raising the possibility that the effect of PD on NO release may be reversible. In later stages of this project it is planned to use dielectric heating to rewarm the tissues and thereby avoid devitrification. The effects of each cryoprotectant on this mode of heating was therefore studied. Gelatin spheres containing vitrifiable concentrations of each cryoprotectant were rewarmed from -60 degrees C in a radiofrequency applicator. Because the uniformity of heating is related to the dielectric properties of the material, these properties were also measured. PD was the most suitable. These physical measurements, combined with the measurements of toxicity and permeability, indicate that PD is the most favorable cryoprotectant of those tested for use in subsequent stages of this study.     

Electric polarization of polyelectrolyte and colloid media: dielectric versus electro-optic approach

The theories of dielectric dispersion and of electric birefringence as a representative of electro-optic methods are considered and it is shown that they both depend in a similar way simply on the real part of the complex electric polarizability of the macromolecules or the particles. The latter also contains the permanent dipole moment. Experimental data on dielectric dispersion, electric birefringence and electric light scattering of strongly elongated, rod-like poly(tetrafluoroethylene) particles are compared and an attempt is made to extend the dielectric dispersion curve to lower frequencies using electric birefringence and electric light scattering data. Further, the experimental data on dielectric dispersion, electric light scattering, electro-orientation and dipolophoresis for the more complicated Escherichia coli particles are compared. Again, the possibility to extend the 10 kHz-100 MHz dielectric dispersion curve down below 1 Hz by using electric light scattering data is examined. The good matching of the dielectric dispersion and electric light scattering frequency curves found in the overlapping frequency range (10 kHz-5 MHz) essentially enhances the chance that dielectric dispersion below 1 MHz is related to alpha dispersion and not to electrode polarization. Thus it is not only possible to obtain additional information on the mechanism of polarization at lower-frequency dielectric dispersion, but also to extend our knowledge about the effective dielectric properties of biological complex fluids to frequencies essentially below 1 MHz. This could be important for the understanding of the effect of low-frequency electromagnetic fields on living matter.     

Thermal phase and excitonic connectivity in fluorescence induction

Chl fluorescence induction (FI) was recorded in sunflower leaves pre-adapted to darkness or low preferentially PSI light, or inhibited by DCMU. For analysis the FI curves were plotted against the cumulative number of excitations quenched by PSII, n _q, calculated as the cumulative complementary area above the FI curve. In the +DCMU leaves n _q was <1 per PSII, suggesting pre-reduction of Q _A during the dark pre-exposure. A strongly sigmoidal FI curve was constructed by complementing (shifting) the recorded FI curves to n _q = 1 excitation per PSII. The full FI curve in +DCMU leaves was well fitted by a model assuming PSII antennae are excitonically connected in domains of four PSII. This result, obtained by gradually reducing Q _A in PSII with pre-blocked Q _B (by DCMU or PQH₂), differs from that obtained by gradually blocking the Q _B site (by increasing DCMU or PQH₂ level) in leaves during (quasi)steady-state e^? transport (Oja and Laisk, Photosynth Res 114, 15–28, 2012). Explanations are discussed. Donor side quenching was characterized by comparison of the total n _q in one and the same dark-adapted leaf, which apparently increased with increasing PFD during FI. An explanation for the donor side quenching is proposed, based on electron transfer from excited P680* to oxidized tyrosine Z (TyrZ^ox). At high PFDs the donor side quenching at the J inflection of FI is due mainly to photochemical quenching by TyrZ^ox. This quenching remains active for subsequent photons while TyrZ remains oxidized, following charge transfer to Q _A. During further induction this quenching disappears as soon as PQ and Q _A become reduced, charge separation becomes impossible and TyrZ is reduced by the water oxidizing complex.     

The permeability of hydrophobic membranes to 22Na salts and 14CO2 in low dielectric media

The one-way fluxes of 14CO2 and a series of 22Na (Cl, Br, HCO3, ClO4, I) salts across n-hexadecane-impregnated solid-support liquid membranes have been measured in water and low dielectric media (50-90 vol% dioxane/water). One-way fluxes for 14CO2 (J14CO2) were 0.84 and 1.03 x 10(-9) mol cm-2 s-1 in 75% dioxane (aq.) and water, respectively, across both impregnated cellulose and teflon membranes. 22Na fluxes across impregnated cellulose membranes in 75% dioxane (aq.) ranged from 1.8 to 11.4 x 10(-10) mol cm-2 s-1 and had the order NaCl less than NaBr less than NaHCO3 less than NaClO4 less than Nal. 22Na fluxes across impregnated teflon membranes were slightly smaller, 1.5-7.1 x 10(-10) mol cm-2 s-1, but had the same order for the anions tested. No measurable 22Na fluxes were observed in aqueous media. For NaI and NaClO4 there was a 3-6-fold enhancement of fluxes in 90% dioxane (aq.) compared to 75% dioxane (aq.). The corresponding enhancement for fluxes of NaHCO3, NaBr and NaCl was 1.5-fold. The results are discussed in terms of ion-paired salt transport in low dielectric media.     

Mechanochemical couplings of kinesin motors

Kinesins are molecular motors capable of moving processively along microtubule in a stepwise manner by hydrolyzing ATP. Numerous experimental results on various aspects of their dynamical behaviours are available in literature. Although a number of models of tightly coordinated mechanism have been proposed to explain some experimental results, up to now no good explanation has been given to all these experimental results by using a single model. We have recently proposed such a model of partially coordinated hand-over-hand moving mechanism. In this paper, we use this model to study in detail various aspects of the dynamical properties of single kinesin molecules. We show that kinesin dimers walk hand-over-hand along microtubules in a partially coordinated rather than a tightly coordinated manner. The degree of coordination depends on the ratio of the two heads' ATPase rates that are in turn determined by both internal elastic force and external load. We have tested this model using various available experimental results on different samples and obtained a good agreement between the theory and the experiments.     

Spin-orbit couplings in the DNA base pairs

The radiative lifetimes of the phosphorescent states of the adenine.thymine (A.T) and guanine.cytosine (G.C) base pairs were calculated on the basis of the singlet-triplet transition probability induced by spin-orbit couplings. The calculated radiative lifetimes averaged over the triplet sublevels of spin state were in the order of G.C less than A.T and in good correlation with those of the composite bases. On the whole the results suggested an important role for thymine triplet having a relatively long lifetime during the course of the triplet localization in DNA, in agreement with the experimental observation that the concentration of triplet is remarkably enhanced with increase in A+T content.     

Residual dipolar couplings in nucleic acid structure determination

Solution NMR spectroscopy of nucleic acids has been limited by the short-range nature of the nuclear Overhauser effect and scalar coupling restraints normally used in structure determination. The addition of residual dipolar couplings, obtained from slightly oriented mixtures, provides bond vector angles relative to a universal alignment tensor. The accurate determination of helix curvature, domain orientation and the stoichiometry of homomultimeric nucleic acid complexes is now possible.     

Determination of residual dipolar couplings in homonuclear MOCCA-SIAM experiments

In solutions with partial molecular alignment, anisotropic magnetic interactions such as the chemical shift anisotropy, the electric quadrupole interaction, and the magnetic dipole-dipole interaction are no longer averaged out to zero in contrast to isotropic solutions. The resulting residual anisotropic magnetic interactions are increasingly used in biological NMR studies for the determination of 3D structures of proteins and other biomolecules. In the present paper we propose a new approach allowing the measurement of residual H^N-H dipolar couplings of non-isotope enriched proteins based on the application of the MOCCA-SIAM experiment. This experiment allows the measurement of homonuclear coupling constants with an accuracy of ca. ±0.2 Hz and is therefore particularly well suited to determine residual dipolar couplings at relatively low degrees of molecular orientation. The agreement between experimentally determined residual H^N-H couplings and calculated values is demonstrated for BPTI.     

Global changes of chlorophyll excitonic interactions in photosystem II during thermal denaturation

Photosystem II (PSII) is a multisubunit chlorophyll-binding enzyme that absorbs light to catalyze water oxidation and plastoquinone reduction. Chlorophyll excitonic interaction changes in PSII were studied by absorption and circular dichroism spectra from 25 degrees C to 80 degrees C, and protein subunit denaturation was monitored by differential scanning calorimetry. A four-stage process of chlorophyll excitonic interaction change was observed being correlated with the denaturation of protein subunits.     

Recognition of protein folds via dipolar couplings

Alignment of proteins in dilute liquid crystalline medium gives rise to residual dipolar couplings which provide orientational information of vectors connecting the interacting nuclei. Considering that proteins are mainly composed of regular secondary structures in a finite number of different mutual orientations, main chain dipolar couplings appear sufficient to reveal structural resemblance. Similarity between dipolar couplings measured from a protein and corresponding values computed from a known structure imply homologous structures. For dissimilar structures the agreement between experimental and calculated dipolar couplings remains poor. In this way protein folds can be readily recognized prior to a comprehensive structure determination. This approach has been demonstrated by showing the similarity in fold between the hitherto unknown structure of calerythrin and sarcoplasmic calcium-binding proteins from Nereis diversicolor and Branchiostoma lanceolatum with known crystal structures.     

Evaluation of uncertainty in alignment tensors obtained from dipolar couplings

Residual dipolar couplings and their corresponding alignment tensors are useful for structural analysis of macromolecules. The error in an alignment tensor, derived from residual dipolar couplings on the basis of a known structure, is determined not only by the accuracy of the measured couplings but also by the uncertainty in the structure (structural noise). This dependence is evaluated quantitatively on the basis of simulated structures using Monte-Carlo type analyses. When large numbers of dipolar couplings are available, structural noise is found to result in a systematic underestimate of the magnitude of the alignment tensor. Particularly in cases where only few dipolar couplings are available, structural noise can cause significant errors in best-fitted alignment tensor values, making determination of the relative orientation of small fragments and evaluation of local backbone mobility from dipolar couplings difficult. An example for the protein ubiquitin demonstrates the inherent limitations in characterizing motions on the basis of local alignment tensor magnitudes.     

Intramolecular vibrations enhance the quantum efficiency of excitonic energy transfer

Photosynthesis Research - We study the impact of underdamped intramolecular vibrational modes on the efficiency of the excitation energy transfer in a dimer in which each state is coupled to its...     

Dielectric increment and dielectric dispersion of solutions containing simple charged linear macromolecules. I. Theory

A theory is derived for the static and frequency dependent value of the electric permittivity for model systems representing a solution of a macromolecule bearing a large number of identical charges. The polyion is represented either as a charged rigid rod (A) or as a sequence of charged rodlike subunits in an arbitrary but fixed configuration (B) and it is assumed that a certain fraction of the counterions is closely associated to the macromolecule. The dielectric properties are described in terms of fluctuations in the distribution of the associated counterions along the polyion. These fluctuations can occur locally between potential barriers marking the ends of the subunits (if considered) but can also extend over the whole molecule. Neglecting correlations between different associated counterions expressions for the static value of the dielectric increment are obtained which reveal its dependence on the fraction of bound ions, on the charge of the counterions and on the length of the molecule for model A or the radius of gyration for model B. The dynamic behaviour of A is distinguishable from that of B as the former will present one single dispersion curve of the frequency dependent electric permittivity while the latter may give rise to two different dispersion regions. This will be the case if both the exchange between bound and free ions and the rotation of the complete molecule are relatively slow in comparison to the local bound counterion density fluctuations and if these fluctuations occur on a much shorter time scale than the ion density fluctuations extending over the complete macromolecule.     

Visualization of excitonic structure in the Fenna-Matthews-Olson photosynthetic complex by polarization-dependent two-dimensional electronic spectroscopy

Photosynthetic light-harvesting proceeds by the collection and highly efficient transfer of energy through a network of pigment-protein complexes. Interchromophore electronic couplings and interactions between pigments and the surrounding protein determine energy levels of excitonic states, and dictate the mechanism of energy flow. The excitonic structure (orientation of excitonic transition dipoles) of pigment-protein complexes is generally deduced indirectly from x-ray crystallography, in combination with predictions of transition energies and couplings in the chromophore site basis. We demonstrate that coarse-grained, excitonic, structural information in the form of projection angles between transition dipole moments can be obtained from the polarization-dependent, two-dimensional electronic spectroscopy of an isotropic sample, particularly when the nonrephasing or free polarization decay signal, rather than the photon echo signal, is considered. This method provides an experimental link between atomic and electronic structure, and accesses dynamical information with femtosecond time resolution. In an investigation of the Fenna-Matthews-Olson complex from green sulfur bacteria, the energy transfer connecting two particular exciton states in the protein was isolated as the primary contributor to a crosspeak in the nonrephasing two-dimensional spectrum at 400 femtoseconds under a specific sequence of polarized excitation pulses. The results suggest the possibility of designing experiments using combinations of tailored polarization sequences to separate and monitor individual relaxation pathways.     

Quantum mechanical analysis of excitation energy transfer couplings in photosystem II

We evaluated excitation energy transfer (EET) coupling (J) between all pairs of chlorophylls (Chls) and pheophytins (Pheos) in the protein environment of photosystem II based on the time-dependent density functional theory with a quantum mechanical/molecular mechanics approach. In the reaction center, the EET coupling between Chls P_D1 and P_D2 is weaker (|J(P_D1/P_D2)| = 79 cm^?1), irrespective of a short edge-to-edge distance of 3.6 Å (Mg-to-Mg distance of 8.1 Å), than the couplings between P_D1 and the accessory Chl_D1 (|J(P_D1/Chl_D2)| = 104 cm^?1) and between P_D2 and Chl_D2 (|J(P_D2/Chl_D1)| = 101 cm^?1), suggesting that P_D1 and P_D2 are two monomeric Chls rather than a “special pair”. There exist strongly coupled Chl pairs (|J| > ～100 cm^?1) in the CP47 and CP43 core antennas, which may be candidates for the red-shifted Chls observed in spectroscopic studies. In CP47 and CP43, Chls ligated to CP47-His26 and CP43-His56, which are located in the middle layer of the thylakoid membrane, play a role in the “hub” that mediates the EET from the lumenal to stromal layers. In the stromal layer, Chls ligated to CP47-His466, CP43-His441, and CP43-His444 mediate the EET from CP47 to Chl_D2/Pheo_D2 and from CP43 to Chl_D1/Pheo_D1 in the reaction center. Thus, the excitation energy from both CP47 and CP43 can always be utilized for the charge-separation reaction in the reaction center.