Monoalkyl phosphodiesters: Synthesis and dielectric relaxation of solutions

Chromatographically pure hexadecylphosphocholine, -(N,N-dimethyl)-ethanolamine, -(N-N-methyl)-ethanolamine and -ethanolamine have been synthesized. Aqueous solutions of these phospholipids have been prepared for the purpose of measuring their dielectric spectra. Micellar solutions appropriate for the dielectric studies were obtained with the choline and the (N,N-dimethyl)-ethanolamine head groups.The dielectric spectra of these phospholipid/water systems are evaluated in terms of phenomenologically introduced sum of Cole-Cole relaxation functions and also on the basis of a model relaxation function which has regard to internal depolarizing fields of the colloidal solutions. Parameters reflecting the motions of the dipolar head groups and of the hydration water molecules of the synthetic monoalkyl phosphodiesters are discussed and are compared with those for egg lysolecithin.The mobility of the dipolar phospholipid head groups and the number of influenced water molecules per zwitterion decreases when changing from lysolecithin to hexadecylphosphocholine and further to hexadecylphospho-(N,N-dimethyl)-ethanolamine, while the relaxation time of the hydration water increases. These results indicate the micellar surface to get less porous within the above series of lipids.     

Solvent proton relaxation studies of cytochrome c oxidase solutions

The interaction of solvent water protons with the bound paramagnetic metal ions of beef heart cytochrome c oxidase has been examined. The observed proton relaxation rates of enzyme solutions had a negative temperature dependence, indicating a rapid exchange between solvent protons in the coordination sphere of the metal ions and bulk solvent. An analysis of the dependence of the proton relaxation rate on the observation frequency indicated that the correlation time, which modulates the interaction between solvent protons and the unpaired electrons on the metal ions, is due to the electron spin relaxation time of the heme irons of cytochrome c oxidase. This means that at least one of the hemes is exposed to solvent. The proton relaxation rate of the oxidized enzyme was found to be sensitive to changes in ionic strength and to changes in the spin states of the metal ions. Heme a3 was found to be relatively inaccessible to bulk solvent. Partial reduction of the enzyme caused a slight increase in the relaxation rate, which may be due to a change in the antiferromagnetic coupling between two of the bound paramagnetic centers. Further reduction resulted in a decreased relaxation rate, and the fully reduced enzyme was no longer sensitive to changes in ionic strength. The binding of cytochrome c to cytochrome c oxidase had little effect on the proton relaxation rates of oxidized cytochrome oxidase indicating that cytochrome c binding has little effect on solvent accessibility to the metal ion sites.     

On the mechanism of dielectric relaxation in aqueous DNA solutions.

The complex dielectric response of calf thymus DNA in aqueous saline solutions has been measured from 1 MHz to 1 GHz. The results are presented in terms of the relaxation of the incremental contributions to the permittivity and conductivity from the condensed counterions surrounding the DNA molecules. Measurements of the low-frequency conductivity of the samples also lends support to the condensed counterion interpretation.     

A proton magnetic relaxation study of ferrimyoglobin in aqueous ionic solutions

Proton magnetic longitudinal T₁ relaxation times have been measured for acid (horse) ferrimyoglobin solutions [0.1 M NaCl and KH₂PO₄, 2 M NaCl and 1 M MgCl₂] from 5°C to 35°C in dependence on myoglobin concentration up to 6 mM. The enhancement of the relaxation rate due to the paramagnetic haem iron. which is observed in this temperature range is compared with analogous data for the ferrihaemoglobin solution. The conclusion is that the protons exchanging from the haem pocket with bulk solvent are not those from the water molecule at the sixth ligand site of haem iron. The exchanging protons are more than 4 Å away from the haem iron being closer to it in ferrimyoglobin than in ferrihaemogiobin. This distance becomes larger in solutions with higher salt concentration, the largest difference between 0.1 M NaCl and 1 M MgCl₂ being over one Angstrom unit. This indicates a conformational change of the haem pocket, possibly its tightening.     

The molecular mechanism of the temperature enhancement of proton magnetic relaxation rates in methaemoprotein solutions

The mechanism of water exchange between the haem-pocket and bulk solvent in aqueous methaemopiotein solutions was firmly substantiated by using the aliphatic protons of certain lower alcohols in an otherwise deuterated solution for measuring the incremental relaxation rates resulting from their magnetic interaction with the haem-iron. The fast-exchange condition was established for solutions of horse fluorometmyoglobin, human A fluoromethaemoglobin and Chironomus thummi aquomethaemoglobin. The distances between the exchangeable protons and the haem-iron obtained from these PMR measurements concur with the presence of the fluoride ion, while for Chironomus aquomethaemoglobin this distance is also much larger than that resulting from the location of the 6th site Water molecule. The latter finding is the first clear-cut evidence that the exchanging protons belong to the next neighbour water molecule, a previously advanced hypothesis. The exchanging water molecule may thus serve as a natural probe for comparing the haem-pocket conformational state(s) under different conditions or in various haemoproteins.     

Frequency dependence of the proton magnetic relaxation in aqueous solutions iof haemoproteins

The longitudinal proton magnetic relaxation times T₁ were measured for ferri (met)-and carbonmonoxy-bovine haemoglobin and equine myoglobin in 0.1 M KH₂PO₄ aqueous solutions near pH 6 at 5°C and 35°C from 1.5- to 60-MHz Larmor frequencies. It is concluded that the correlation time τ_C for the dipole–dipole interaction of electron and nuclear spins is in fact the electron (ferric) spin relaxation time τ_S being close to 1.5 × 10^?10 sec for both metHb and metMb at 5°C. At 35°C the paramagnetic relaxation rates are not determined solely by the relaxation of protons exchanging from the haem pocket with bulk solvent. Hence, τC at 35°C cannot be calculated from the dispersion data obtained at this temperature. The relevance of this for the determination of interspin distances r is discussed.     

Dynamics of protein conformational fluctuation in enzyme catalysis with special attention to proton transfers in serine proteinases

We have provided a quantum mechanical model for proteinase-catalyzed peptide, amide and ester hydrolysis. The model rests on electron and atom transfer theory, but incorporates the dynamics of conformational nuclear modes as a new element. The model is applied to acylation, but can straightaway be extended to deacylation, and is substantiated by recent structural and kinetic data for proteinase enzyme catalysis. The role of the conformational modes is found to be two-fold. First, the crystallographic distances for the proton transfers involved are far too large for direct transfer. His-57 mobility, handled stochastically, to bring the donor and acceptor groups within suitable reach, is therefore a crucial element of the theory. Secondly, the charge alignment in the Asp-102/His-57/tetrahedral intermediate system implies that the curvature of the potential surface along the conformational coordinates in this state is much lower than in the initial enzyme-substrate and final acyl states. A consequence of this is that the activation energy liberated after the first proton transfer is not dissipated, but stored in the conformational system and used in the second proton transfer step.     

The effect of protein relaxation on charge-charge interactions and dielectric constants of proteins.

The effect of the reorganization of the protein polar groups on charge-charge interaction and the corresponding effective dielectric constant (epsilon(eff)) is examined by the semimicroscopic version of the Protein Dipole Langevin Dipoles (PDLD/S) method within the framework of the Linear Response Approximation (LRA). This is done by evaluating the interactions between ionized residues in the reaction center of Rhodobacter sphaeroides, while taking into account the protein reorganization energy. It is found that an explicit consideration of the protein relaxation leads to a significant increase in epsilon(eff) and that semimicroscopic models that do not take this relaxation into account force one to use a large value for the so-called "protein dielectric constant," epsilon(p), of the Poisson-Boltzmann model or for the corresponding epsilon(in) in the PDLD/S model. An additional increase in epsilon(eff) is expected from the reorganization of ionized residues and from changes in the degree of water penetration. This finding provides further support for the idea that epsilon(in) (or epsilon(p)) represents contributions that are not considered explicitly. The present study also provides a systematic illustration of the nature of epsilon(eff), supporting our previously reported view that charge-charge interactions correspond to a large value of this "dielectric constant," even in protein interiors. It is also pointed out that epsilon(eff) for the interaction between ionizable groups in proteins is very different from the effective dielectric constant, epsilon'(eff), that determines the free energy of ion pairs in proteins (epsilon'(eff) reflects the effect of preoriented protein dipoles). Finally, the problems associated with the search for a general epsilon(in) are discussed. It is clarified that the epsilon(in) that reproduces the effect of protein relaxation on charge-charge interaction is not equal to the epsilon(in) that reproduces the corresponding effect upon formation of individual charges. This reflects fundamental inconsistencies in attempts to cast microscopic concepts in a macroscopic model. Thus one should either use a large epsilon(in) for charge-charge interactions and a small epsilon(in) for charge-dipole interactions or consider the protein relaxation microscopically.     

Trehalose effect on low temperature protein dynamics: fluctuation and relaxation phenomena

We performed spectral diffusion experiments in trehalose-enriched glycerol/buffer-glass on horseradish peroxidase where the heme was replaced by metal-free mesoporphyrin IX, and compared them with the respective behavior in a pure glycerol/buffer-glass (Schlichter et al., J. Chem. Phys. 2000, 112:3045-3050). Trehalose has a significant influence: spectral diffusion broadening speeds up compared to the trehalose-free glass. This speeding up is attributed to a shortening of the correlation time of the frequency fluctuations most probably by preventing water molecules from leaving the protein interior. Superimposed to the frequency fluctuation dynamics is a relaxation dynamics that manifests itself as an aging process in the spectral diffusion broadening. Although the trehalose environment speeds up the fluctuations, it does not have any influence on the relaxation. Both relaxation and fluctuations are governed by power laws in time. The respective exponents do not seem to change with the protein environment. From the spectral dynamics, the mean square displacement in conformation space can be determined. It is governed by anomalous diffusion. The associated frequency correlation time is incredibly long, demonstrating that proteins at low temperatures are truly nonergodic systems.     

Counterion fluctuation and dielectric dispersion in linear polyelectrolytes

The thermal fluctuation in the concentration of counterions bound to a rodlike polyion was analyzed by expanding the fluctuation in a Fourier series along the rod. The amplitude and the relaxation time of fluctuations of various wave lengths were obtained as functions of the charge density and the length of the polyion. From these results the real and imaginary parts of the dielectric constant of the polyelectrolyte solution were derived as the sum of contributions of fluctuations of different modes. The dielectric dispersion curve or the Cole-Cole plot obtained was found to be in good agreement with experimental data.     

NMR relaxation of protein and water protons in diamagnetic hemoglobin solutions

We have measured T1 and T2 of protein and water protons in hemoglobin solutions using broad-line pulse techniques; selective excitation and detection methods enabled the intrinsic protein and water relaxation rates, as well as the spin-transfer rate between them, to be obtained at 5, 10, and 20 MHz. Water and protein T1 data were also obtained at 100 and 200 MHz for hemoglobin in H2O/D2O mixtures by using commercial Fourier-transform instruments. The T1 data conform to a simple model of two well-mixed spin systems with single intrinsic relaxation times and an average spin-transfer rate, with each phase recovering from a radio-frequency excitation with a biexponential time dependence. At low frequencies, protein T1 and T2 agree reasonably with a model of dipolar relaxation of an array of fixed protons tumbling in solution, explicitly calculating methyl and methylene relaxation and using a continuum approximation for the others. Differing values in H2O and D2O are mainly ascribed to solvent viscosity. For water-proton relaxation, T1, T2, and spin transfer were measured for H2O and HDO, which enabled a separation of inter-and intramolecular contributions to relaxation. Despite such detail, few firm conclusions could be reached about hydration water. But it seems clear that few long-lived hydration sites are needed to explain T1 and T2, and the spin-transfer value mandates fewer than five sites with a lifetime longer than 10(-8) s.     

The effect of low molecular weight ligands on relaxation of water protons in protein solutions

Interaction of low-molecular ligands (LML) isolated from blood serum albumin (SA) and serum proteins leads to higher T1 values for water protons compared with those observed in LML-free solutions, although the total amount of bound water increases. The latter was revealed by low-temperature NMR spectroscopy, as well as by the amount of water sorbed on SA + LML at a relative humidity P/Ps greater than 0.7. In the region 0.2 less than P/Ps less than 0.6 the amount of SA + LML-sorbed water decreased, as compared with that in SA indicating that the oppositely charged groups of LML screen some charged groups of the protein. A decrease of charge-to-charge interactions in solution, or with a high water content, leads to the hydration of those groups. The increase of the T1 value for water protons in solution is, probably, due to a hindered exchange between the sorbed water and bulk water. It is outlined that charge interactions between macromolecules may significantly affect water sorption by proteins.     

The contribution of mitochondrial calcium ion exchange to relaxation of tension in cardiac muscle

The possible contribution of mitochondrial Ca²⁺ accumulation and release to contractile phenomena has been investigated. Two intracellular fractions of Ca²⁺ sequestration can be identified in cardiac myocytes, one ascribed to mitochondria. Two modes of Ca²⁺ transport exist within the mitochondrial fraction, one dependent upon mitochondrial respiration and the other upon extramitochondrial [Na⁺]. Experiments with trabeculae show that under appropriate conditions, the rate of relaxation and the amount of tension developed is dependent on these two modes of Ca²⁺ transport. A model is presented quantifying the contribution of the mitochondria to relaxation.     

A proton magnetic relaxation study of ferric myoglobin and haemoglobin in water/ethanediol solutions.

Structural alterations of the haem vicinity of the high-spin derivatives of bovine ferric myoglobin (metmyoglobin) and human haemoglobin and the changes of the interaction with inositol hexaphosphate induced by ethanediol were monitored by solvent-proton magnetic relaxation. On addition of ethanediol up to 60% the fluoromet derivatives exhibit a gradual increase in the accessibility of the haem for the molecules from the solvent. In aquomethaemoglobin solutions with more than 25% ethanediol there is no unique explanation of proton magnetic relaxation. Ethanediol enhances the binding of inositol hexaphosphate to methaemoglobin, but the structural consequences of this binding on the haem-pockets seem to be diminished. The mechanisms of the observed structural and functional alterations of myoglobin as well as haemoglobin tetramer are discussed here.     

Measurement of proton relaxation rates in proteins

Summary Five different types of experiment are described which make it possible to measure various relaxation rates of selected protons in crowded spectra of macromolecules such as proteins: longitudinal spin-lattice relaxation rates =1/T₁, transverse relaxation rates =1/T₂ measured under conditions of free precession, transverse relaxation rates ¹ _LOCK=1/T₁ measured under conditions of spin-locking, and transverse relaxation rates ^DQC=1/T₂ ^DQC and ^ZQC=1/T₂ ^ZQC of double- and zero-quantum coherences. The surprisingly large discrepancy between the transverse rates ^t and ^t is discussed in detail. To separate overlapping proton signals, the experimental schemes involve one or several magnetization transfer steps, using a doubly selective homonuclear Hartmann-Hahn method. Numerous variants of the basic ideas can be conceived, depending on the extent of signal overlap and on the topology of the networks of scalar couplings. Applications are shown to H and H of Tyr²³, to H, H and H of Cys³⁰, and to H and H of Ala²⁴ in bovine pancreatic trypsin inhibitor (BPTI).     

The water proton spin-lattice relaxation times in virus-infected cells.

The water proton spin-lattice relaxation times in HEp-2 cell cultures were determined immediately after 1 h of polio-virus adsorption. The shortening of the water T1 was closely related to the multiplicity of infection, allowing direct inspections of the virus--cell interaction since the first steps of the infectious cycle. Virus-induced structural and conformational changes of cell constituents were suggested to be detectable by NMR investigation of cell water.