Cooperative properties of lipid bilayers : collective director fluctuations and the effects of hydrophobic mismatch in protein/lipid membrane systems

Cooperative director fluctuations in lipid bilayers have been postulated for many years. ^2H-NMR T_1^(-1), T_(1P)^(-1) , and T_2^(-1); measurements have been used identify these motions and to determine the origin of increased slow bilayer motion upon addition of unlike lipids or proteins to a pure lipid bilayer.

The contribution of cooperative director fluctuations to NMR relaxation in lipid bilayers has been expressed mathematically using the approach of Doane et al.^1 and Pace and Chan.^2 The T_2^(-1)’s of pure dimyristoyllecithin (DML) bilayers deuterated at the 2, 9 and 10, and all positions on both lipid hydrocarbon chains have been measured. Several characteristics of these measurements indicate the presence of cooperative director fluctuations. First of all, T_2^(-1) exhibits a linear dependence on S2/CD. Secondly, T_2^(-1) varies across the ^2H-NMR powder pattern as sin^2 (2, β), where , β is the angle between the average bilayer director and the external magnetic field. Furthermore, these fluctuations are restricted near the lecithin head group suggesting that the head group does not participate in these motions but, rather, anchors the hydrocarbon chains in the bilayer.

T_2^(-1)has been measured for selectively deuterated liquid crystalline DML hilayers to which a host of other lipids and proteins have been added. The T_2^(-1) of the DML bilayer is found to increase drastically when chlorophyll a (chl a) and Gramicidin A' (GA') are added to the bilayer. Both these molecules interfere with the lecithin head group spacing in the bilayer. Molecules such as myristic acid, distearoyllecithin (DSL), phytol, and cholesterol, whose hydrocarbon regions are quite different from DML but which have small,neutral polar head groups, leave cooperative fluctuations in the DML bilayer unchanged.

The effect of chl a on cooperative fluctuations in the DML bilayer has been examined in detail using ^2H-NMR T_1^(-1), T_(1P)^(-1) , and T_2^(-1); measurements. Cooperative fluctuations have been modelled using the continuum theory of the nematic state of liquid crystals. Chl a is found to decrease both the correlation length and the elastic constants in the DML bilayer.

A mismatch between the hydrophobic length of a lipid bilayer and that of an added protein has also been found to change the cooperative properties of the lecithin bilayer. Hydrophobic mismatch has been studied in a series GA' / lecithin bilayers. The dependence of 2H-NMR order parameters and relaxation rates on GA' concentration has been measured in selectively deuterated DML, dipalmitoyllecithin (DPL), and DSL systems. Order parameters, cooperative lengths, and elastic constants of the DML bilayer are most disrupted by GA', while the DSL bilayer is the least perturbed by GA'. Thus, it is concluded that the hydrophobic length of GA' best matches that of the DSL bilayer. Preliminary Raman spectroscopy and Differential Scanning Calorimetry experiments of GA' /lecithin systems support this conclusion. Accommodation of hydrophobic mismatch is used to rationalize the absence of H_(II) phase formation in GA' /DML systems and the observation of H_(II) phase in GA' /DPL and GA' /DSL systems.

1. J. W. Doane and D. L. Johnson, Chem. Phy3. Lett., 6, 291-295 (1970). 2. R. J. Pace and S. I. Chan, J. Chem. Phy3., 16, 4217-4227 (1982).

Mechanistic studies of a model reaction for enzymatic hydroxylation

A study has been made of the reaction mechanism of a model system for enzymatic hydroxylation. The results of a kinetic study of the hydroxylation of 2-hydroxyazobenzene derivatives by cupric ion and hydrogen peroxide are presented. An investigation of kinetic orders indicates that hydroxylation proceeds by way of a coordinated intermediate complex consisting of cupric ion and the mono anions of 2-hydroxyazobenzene and hydrogen peroxide. Studies with deuterated substrate showed the absence of a primary kinetic isotope effect and no evidence of an NIH shift. The effect of substituents on the formation of intermediate complexes and the overall rate of hydroxylation was studied quantitatively in aqueous solution. The combined results indicate that the hydroxylation step is only slightly influenced by ring substitution. The substituent effect is interpreted in terms of reaction by a radical path or a concerted mechanism in which the formation of ionic intermediates is avoided. The reaction mechanism is discussed as a model for enzymatic hydroxylation.

I. Electronic states of heavily-doped molecular crystals--naphthalene. I. Theoretical. II. Experimental. II. Lowest singlet and triplet excited states of pyrazine

PART I

The energy spectrum of heavily-doped molecular crystals was treated in the Green’s function formulation. The mixed crystal Green’s function was obtained by averaging over all possible impurity distributions. The resulting Green’s function, which takes the form of an infinite perturbation expansion, was further approximated by a closed form suitable for numerical calculations. The density-of-states functions and optical spectra for binary mixtures of normal naphthalene and deuterated naphthalene were calculated using the pure crystal density-of-state functions. The results showed that when the trap depth is large, two separate energy bands persist, but when the trap depth is small only a single band exists. Furthermore, in the former case it was found that the intensities of the outer Davydov bands are enhanced whereas the inner bands are weakened. Comparisons with previous theoretical calculations and experimental results are also made.

PART II

The energy states and optical spectra of heavily-doped mixed crystals are investigated. Studies are made for the following binary systems: (1) naphthalene-h₈ and d₈, (2) naphthalene--h₈ and αd₄, and (3) naphthalene--h₈ and βd₁, corresponding to strong, medium and weak perturbations. In addition to ordinary absorption spectra at 4˚K, band-to-band transitions at both 4˚K and 77˚K are also analyzed with emphasis on their relations to cooperative excitation and overall density-of-states functions for mixed crystals. It is found that the theoretical calculations presented in a previous paper agree generally with experiments except for cluster states observed in system (1) at lower guest concentrations. These features are discussed semi-quantitatively. As to the intermolecular interaction parameters, it is found that experimental results compare favorably with calculations based on experimental density-of-states functions but not with those based on octopole interactions or charge-transfer interactions. Previous experimental results of Sheka and the theoretical model of Broude and Rashba are also compared with present investigations.

PART III

The phosphorescence, fluorescence and absorption spectra of pyrazine-h₄ and d₄ have been obtained at 4˚K in a benzene matrix. For comparison, those of the isotopically mixed crystal pyrazine-h₄ in d₄ were also taken. All these spectra show extremely sharp and well-resolved lines and reveal detailed vibronic structure.

The analysis of the weak fluorescence spectrum resolves the long-disputed question of whether one or two transitions are involved in the near-ultraviolet absorption of pyrazine. The “mirror-image relationship” between absorption and emission shows that the lowest singlet state is an allowed transition, properly designated as ¹B_3u ← ¹A_1g. The forbidden component ¹B_2g, predicted by both “exciton” and MO theories to be below the allowed component, must lie higher. Its exact location still remains uncertain.

The phosphorescence spectrum when compared with the excitation phosphorescence spectra, indicates that the lowest triplet state is also symmetry allowed, showing a strong 0-0 band and a “mirror-image relationship” between absorption and emission. In accordance with previous work, the triplet state is designated as ³B_3u.

The vibronic structure of the phosphorescence spectrum is very complicated. Previous work on the analysis of this spectrum all concluded that a long progression of v_6a exists. Under the high resolution attainable in our work, the supposed v_6a progression proves to have a composite triplet structure, starting from the second member of the progression. Not only is the v_9a hydrogen-bending mode present as shown by the appearance of the C-D bending mode in the d₄ spectrum, but a band of 1207 cm^-1 in the pyrazine in benzene system and 1231 cm^-1 in the mixed crystal system is also observed. This band is assigned as 2v_6b and of a_1g symmetry. Its anonymously strong intensity in the phosphorescence spectrum is interpreted as due to the Fermi resonance with the 2v_6a and v_9a band.

To help resolve the present controversy over the crystal phosphorescence spectrum of pyrazine, detailed vibrational analyses of the emission spectra were made. The fluorescence spectrum has essentially the same vibronic structure as the phosphorescence spectrum.

I. The crystal structure of potassium bis(tricyanovinyl)amine. II. Nuclear magnetic resonance studies of alkali halide solutions. III. Elucidation of the intramolecular hydrogen bond in acetylacetone by the deuterium isotope effect on the chemical shift of the bridge hydrogen

Part I

Potassium bis-(tricyanovinyl) amine, K⁺N[C(CN)=C(CN)₂]₂^-, crystallizes in the monoclinic system with the space group Cc and lattice constants, a = 13.346 ± 0.003 Å, c = 8.992 ± 0.003 Å, B = 114.42 ± 0.02°, and Z = 4. Three dimensional intensity data were collected by layers perpendicular to b* and c* axes. The crystal structure was refined by the least squares method with anisotropic temperature factor to an R value of 0.064.

The average carbon-carbon and carbon-nitrogen bond distances in –C-CΞN are 1.441 ± 0.016 Å and 1.146 ± 0.014 Å respectively. The bis-(tricyanovinyl) amine anion is approximately planar. The coordination number of the potassium ion is eight with bond distances from 2.890 Å to 3.408 Å. The bond angle C-N-C of the amine nitrogen is 132.4 ± 1.9°. Among six cyano groups in the molecule, two of them are bent by what appear to be significant amounts (5.0° and 7.2°). The remaining four are linear within the experimental error. The bending can probably be explained by molecular packing forces in the crystals.

Part II

The nuclear magnetic resonance of ⁸¹Br and ¹²⁷I in aqueous solutions were studied. The cation-halide ion interactions were studied by studying the effect of the Li⁺, Na⁺, K⁺, Mg⁺⁺, Cs⁺ upon the line width of the halide ions. The solvent-halide ion interactions were studied by studying the effects of methanol, acetonitrile, and acetone upon the line width of ⁸¹Br and ¹²⁷I in the aqueous solutions. It was found that the viscosity plays a very important role upon the halide ions line width. There is no specific cation-halide ion interaction for those ions such as Mg⁺⁺, Di⁺, Na⁺, and K⁺, whereas the Cs⁺ - halide ion interaction is strong. The effect of organic solvents upon the halide ion line width in aqueous solutions is in the order acetone ˃ acetonitrile ˃ methanol. It is suggested that halide ions do form some stable complex with the solvent molecules and the reason Cs⁺ can replace one of the ligands in the solvent-halide ion complex.

Part III

An unusually large isotope effect on the bridge hydrogen chemical shift of the enol form of pentanedione-2, 4(acetylacetone) and 3-methylpentanedione-2, 4 has been observed. An attempt has been made to interpret this effect. It is suggested from the deuterium isotope effect studies, temperature dependence of the bridge hydrogen chemical shift studies, IR studies in the OH, OD, and C=O stretch regions, and the HMO calculations, that there may probably be two structures for the enol form of acetylacetone. The difference between these two structures arises mainly from the electronic structure of the π-system. The relative population of these two structures at various temperatures for normal acetylacetone and at room temperature for the deuterated acetylacetone were calculated.