Addition of hydrophilic and lipophilic compounds of biological relevance to the monoolein/water system. I. Phase behavior

The solubilization of hydrophilic and lipophilic molecules, with biological relevance, in the monoolein/water (MO/W) system has been investigated for phase behavior. Small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) and optical microscopy (OM) have been used to characterize the microstructure of the liquid crystalline phases. Partial phase diagrams of the MO/W system in the presence of sodium decanoate, 1-adamantanamine hydrochloride, decanoic and dodecanoic acids, acetyl salicilic acid and retinol have been determined. The stability of the various phases has been followed for at least eight months. The polarity and the molecular structure of the additive determine whether it is located at the polar interface or in the apolar region of the lipid layer. Therefore, the additive affects the interfacial curvature of the lipid layer differently, which in turn will trigger transition to disparate phases. A cubic-to-reverse hexagonal phase transition has been observed with time for most of the ternary systems, with the exception of 1-adamantanamine hydrochloride and retinol. The release of free glycerol and oleic acid due to MO hydrolysis has been clearly demonstrated by 13C NMR. This would account for the changes in phase behavior observed with time. The released oleic acid, located in the MO acyl chain region, favors the inverse interfacial curvature. The average lipid dimensions in the cubic and in the reverse hexagonal phases have been calculated from SAXS data.     

Mobility of water bound to biological membranes: A proton NMR relaxation study

Water proton nuclear magnetic resonance relaxation measurements have been obtained for aqueous suspensions of red cell membranes. These data support a model in which water molecules are exchanging rapidly between a bound phase with restricted motions and a free phase with dynamic properties similar to liquid water. From this model and these data, estimates are obtained for the relaxation time for bound phase water. Possible relaxation mechanisms for bound phase water are discussed and some support is found for an intermolecular interaction modulated by translational motions characterized by a diffusion constant of 10^?9 cm²/s.     

Mobility of water bound to biological membranes. A proton NMR relaxation study.

Water proton nuclear magnetic resonance relaxation measurements have been obtained for aqueous suspensions of red cell membranes. These data support a model in which water molecules are exchanging rapidly between a bound phase with restricted motions and a free phase with dynamic properties similar to liquid water. From this model and these data, estimates are obtained for the relaxation time for bound phase water. Possible relaxation mechanisms for bound phase water are discussed and some support is found for an intermolecular interaction modulated by translational motions characterized by a diffusion constant of 10(-9) cm2/s.     

13C NMR relaxation and conformational flexibility of the deoxyribose ring.

13C T1's and NOE's have been measured for all protonated carbons of 2'-deoxy-D-ribose (2'-d-ribose), 2'-deoxyadenosine-5'-monophosphate (5'-dAMP), thymidine-3'-monophosphate (3'-TMP) and thymidine-5'-monophosphate (5'-TMP) in D2O solutions. In all of the deoxy sugars examined, NT1 values for C-2' are significantly larger than the values for the remaining carbons. This result is interpreted in terms of rapid puckering motion of C-2'. By contrast, NT1 values measured in ribose are found to be equal, within experimental error. The results are compared with analogous data obtained for the five membered pyrrolidine ring of proline and with results for DNA itself.     

Using simple 13C NMR linewidth and relaxation measurements to make detailed chemical shift assignments in triacylglycerols and related compounds

Two simple experiments measuring the ¹³C linewidths ν_1/2 and spin–lattice relaxation times T₁ of each of the signals in the spectrum of trilinolein indicate that the ν_1/2 and T₁ values are consistent with the different degrees of motional freedom expected for the various ¹³C nuclei. However, for each chain, the ν_1/2 and T₁ measurements indicate a small reversal in mobility at C-10 relative to C-9 before motional freedom again steadily increases on each chain starting at C-11. The T₁ experiment allows unambiguous assignments of the C-8 signal and C-14 signal, which differ by only 0.010 ppm. Measurements of ¹³C ν_1/2 and T₁ values on tripalmitin provide secure assignments for the C-5 and C-6 signals, for which conflicting assignments have been reported. The T₁ measurements also show that among the tightly clustered C-8 through C-12 signals, the C-11 signals are the most downfield, while the C-12 signals are the most upfield, again contrary to a previous report. Similar measurements of ¹³C ν_1/2 and T₁ values on other triacylglycerols or related compounds may prove equally useful in making chemical shift assignments and detecting any discontinuities in motional freedom along a chain. The benefits and possible limitations of ultrahigh field NMR for studying triacylglycerols and related compounds are discussed.     

A 13C NMR study of [5,8-13C2]spermidine binding to tRNA and to Escherichia coli macromolecules

[5,8-13C2]Spermidine was prepared by synthesis, and its binding to macromolecular structures of Escherichia coli was studied. When added to E. coli cells, the two signals of [13C]spermidine (C-5, 47.8 ppm, and C-8, 39.6 ppm; JC-C = 5.8 Hz) were strongly broadened due to binding to macromolecules. When [13C]spermidine was added to E. coli tRNA, the C-5 resonance broadened to v1/2 = 4.7 Hz, whereas the C-8 resonance broadened to v1/2 = 2.7 Hz. tRNA-bound [13C]spermidine could be chased by [12C]spermidine or spermine, but not by putrescine or cadaverine. By using mixtures of [5-13C]- and [8-13C]spermidines (where 13C-13C coupling was avoided), it was possible to estimate a dissociation constant (Kd) of 3 x 10(-3) M using the C-5 v1/2obs values and a Kd of 2.10(-3) M using the C-8 v1/2obs values. The number of spermidine-binding sites (n) could also be estimated by fitting the bound spermidine molar fraction versus tRNA concentration. Values of n = 12 +/- 2 and 14 +/- 3 were obtained for C-5 and C-8, respectively. Measurements of line narrowing at increasing Mg2+ concentrations indicated that approximately 11 spermidines (of the 12-14 bound ones) could be displaced by the former, whereas 3 spermidines remain strongly bound to the tRNA backbone. Measurements of free and bound T1 allowed the determination of a correlation time of 10(-10)s for tRNA-bound spermidine.     

A 13C NMR spin-lattice relaxation study of the interaction of myelin proteins with lipid vesicles

Natural abundance 13C nuclear magnetic spin-lattice relaxation times have been measured for bovine brain phosphatidylserine vesicles with and without bound proteins. The relaxation times were lower than published values for the corresponding nuclei in egg phosphatidylcholine, but showed the same trend, with relaxation times increasing along the acyl chains away from the polar headgroup. These times were inversely related to the degree of saturation of the lipid. Cytochrome c caused insignificant changes in the lipid acyl chain relaxation rates but reduced the resonance intensities, in agreement with Brown and Wüthrich (Biochem. Biophys. Acta 468 (1977) 389). In contrast, the basic protein from bovine myelin did not affect the intensities but reduced the relaxation times for 13C nuclei near the bilayer centre, and for nuclei near carbon-carbon double bonds. These proteins also dramatically broadened the serine headgroup carboxyl resonance. It appears, in accord with other recent evidence, that the basic protein does penetrate the hydrophobic region of the bilayer (possibly to the centre), producing quantitatively similar changes in the relaxation rates to proteolipid protein, an integral membrane protein.     

NMR studies of dynamics in RNA and DNA by 13C relaxation

RNA and DNA molecules experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. The many functions of RNA in biology very often require this molecule to change its conformation in response to biological signals in the form of small molecules, proteins or other nucleic acids, whereas local motions in DNA may facilitate protein recognition and allow enzymes acting on DNA to access functional groups on the bases that would otherwise be buried in Watson-Crick base pairs. Although these statements make a compelling case to study the sequence dependent dynamics in nucleic acids, there are few residue-specific studies of nucleic acid dynamics. Fortunately, NMR studies of dynamics of nucleic acids and nucleic acids-protein complexes are gaining increased attention. The aim of this review is to provide an update of the recent progress in studies of nucleic acid dynamics by NMR based on the application of solution relaxation techniques.     

13C NMR relaxation times of hepatic glycogen in vitro and in vivo

The field dependence of relaxation times of the C-1 carbon of glycogen was studied in vitro by natural-abundance 13C NMR. T1 is strongly field dependent, while T2 does not change significantly with magnetic field. T1 and T2 were also measured for rat hepatic glycogen enriched with [1-13C]glucose in vivo at 4.7 T, and similar relaxation times were observed as those obtained in vitro at the same field. The in vitro values of T1 were 65 +/- 5 ms at 2.1 T, 142 +/- 10 ms at 4.7 T, and 300 +/- 10 ms at 8.4 T, while T2 values were 6.7 +/- 1 ms at 2.1 T, 9.4 +/- 1 ms at 4.7 T, and 9.5 +/- 1 ms at 8.4 T. Calculations based on the rigid-rotor nearest-neighbor model give qualitatively good agreement with the T1 field dependence with a best-fit correlation time of 6.4 X 10(-9) s, which is significantly smaller than tau M, the estimated overall correlation time for the glycogen molecule (ca. 10(-5) s). A more accurate fit of T1 data using a modified Lipari and Szabo approach indicates that internal fast motions dominate the T1 relaxation in glycogen. On the other hand, the T2 relaxation is dominated by the overall correlation time tau M while the internal motions are almost but not completely unrestricted.     

Use of 31P and 13C NMR to study enzyme mechanisms

A number of complex biochemical problems have been solved recently by application of new techniques in which 31P and 13C NMR spectroscopy is used. Oxygen isotope exchange phenomena were studied by these NMR methods and used to analyze individual mechanistic events in enzymatic reactions. The existence of intermediates in the reactions catalyzed by glutamine synthetase (EC 6.3.1.2) and carbamyl-phosphate synthetase (EC 2.7.2.9) has been established as well as the kinetic competence of these intermediates for each enzyme. The NMR theory and kinetic experiments required to conduct such studies are discussed.     

13C NMR spectra of some naturally occurring binaphthoquinones and related compounds

¹³C NMR chemical shift assignments have been shown to be diagnostic for the establishment of the dimeric linkage of some naturally occurring binaphthoquinones. The unsymmetric ¹³C and ¹H spin-spin coupled pattern observed in the ¹H coupled ¹³C NMR spectrum of plumbagin for C-6 has also been noticed earlier with the related compound juglone. The nature of these effects has been substantiated for the first time using benzene induced solvent shifts and D₂O exchange. ¹³C chemical shift assignments of plumbagin reported earlier for C-6 and C-8 have been revised.     

Application of 13C and 31P NMR to the study of hepatic metabolism

Alternate scan 13C and 31P NMR has been used to follow the metabolism of 13C-labeled substrates, in the presence and absence of insulin, in isolated perfused liver from fasted rats. Because both 31P and 13C NMR spectra are recorded almost simultaneously with this method, both phosphate metabolites and 13C-labeled metabolites are measured, noninvasively and repetitively, to give an immediate, broad survey of the hepatic response to a variety of stimuli. During the metabolism of [2-13C]pyruvate, [1,2-13C]ethanol, and NH4+, 13C-labeled glycogen increases synchronously with, and at the same rate as, the synthesis of 13C-labeled glucose; thus, glycogenesis was essentially a gluconeogenic process under our conditions and was unaltered by the presence of insulin. From the position of the 13C-labeled citrate peak observed in liver, the measurement of KD for the citrate-magnesium complex under our conditions, and the expression relating these quantities to the concentration of free Mg2+, the intracellular level of free Mg2+ is estimated to be 0.46 +/- 0.05 mM. Later administration of glucagon led to a rapid decrease in glycogen and citrate and a 44% increase in glycero-3-phosphocholine (GPC); increase in GPC is consistent with stimulation of liver phospholipase activity by glucagon. Simultaneous administration of two different 13C-labeled substrates, or one doubly labeled substrate, introduced multiplet structure arising from spin-spin interaction between labeled adjacent carbons into the peaks of several key metabolites. The 13C NMR intensity distributions within the several multiplets are used, within the context of a first-order model for fluxes into the Krebs cycle, to estimate relative fluxes under the conditions of the experiment.     

Cytosine ribose flexibility in DNA: a combined NMR 13C spin relaxation and molecular dynamics simulation study

Using (13)C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C-H vectors in all cytosine ribose moieties within the Dickerson-Drew dodecamer (5'-CGCGAATTCGCG-3') are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase.     

Permeability and damage of the erythrocyte membrane at -1 degree C to -9 degrees C shown by the NMR relaxation method

Amount of inter--and extracellular unfrozen water in the intact human erythrocytes as well as tau a, "a life time" of water molecules in these cells, have been determined. The progressive compression of the erythrocytes during freezing them as deep as -5 degrees C has been found to submit to the ideal osmometer law. The membrane impermeability to Mn2+ ions has been disturbed when the temperature falls to -7 degrees C and below, the erythrocytes reaching "the minimal volume".     

Internal mobility of cyclic RGD hexapeptides studied by 13C NMR relaxation and the model-free approach

Summary The internal mobility of three isomeric cyclic RGD hexapeptides designed to contain two -turns in defined positions, cyclo(Arg-Gly-Asp-Gly-d-Pro-Pro) (I), cyclo(Arg-Gly-Asp-d-Pro-Gly-Pro) (II) and cyclo(Arg-Gly-Asp-d-Pro-Pro-Gly) (III), have been studied by ¹³C NMR longitudinal and transverse relaxation experiments and measurements of steady-state heteronuclear {¹H}-¹³C NOE enhancement with ¹³C at natural abundance. The data were interpreted according to the model-free formalism of Lipari and Szabo, which is usually applied to data from macromolecules or larger sized peptides with overall rotational correlation times exceeding 1 ns, to yield information about internal motions on the 10–100 ps time scale. The applicability of the model-free analysis with acceptable uncertainties to these small peptides, with overall rotational correlation times slightly below 0.3 ns, was demonstrated for this specific instance. Chemical exchange contributions to T₂ from slower motions were also identified in the process. According to the order parameters obtained for its backbone -carbon atoms, II has the most rigid backbone conformation on the 10–100 ps time scale, and I the most flexible. This result coincides with the results of earlier NMR-constrained conformational searches, which indicated greatest uncertainty in the structure of I and least in II.     

Insight into the binding of antifreeze proteins to ice surfaces via 13C spin lattice relaxation solid-state NMR

The primary sequences of type I antifreeze proteins (AFPs) are Ala rich and contain three 11-residue repeat units beginning with threonine residues. Their secondary structures consist of alpha-helices. Previous activity study of side-chain mutated AFPs suggests that the ice-binding side of type I AFPs comprises the Thr side chains and the conserved i + 4 and i + 8 Ala residues, where i indicates the positions of the Thrs. To find structural evidence for the AFP's ice-binding side, a variable-temperature dependent (13)C spin lattice relaxation solid-state NMR experiment was carried out for two Ala side chain (13)C labeled HPLC6 isoforms of the type I AFPs each frozen in H(2)O and D(2)O, respectively. The first one was labeled on the equivalent 17th and 21st Ala side chains (i + 4, 8), and the second one on the equivalent 8th, 19th, and 30th Ala side chains (i + 6). The two kinds of labels are on the opposite sides of the alpha-helical AFP. A model of Ala methyl group rotation/three-site rotational jump combined with water molecular reorientation was tested to probe the interactions of the methyl groups with the proximate water molecules. Analysis of the T(1) data shows that there could be 10 water molecules closely capping an i + 4 or an i + 8 methyl group within the range of van der Waals interaction, whereas the surrounding water molecules to the i + 6 methyl groups could be looser. This study suggests that the side of the alpha-helical AFP comprising the i + 4 and i + 8 Ala methyl groups could interact with the ice surface in the ice/water interface.     

Characterization of hydrophilic and lipophilic pathways of Hedera helix L. cuticular membranes: permeation of water and uncharged organic compounds

The permeability of astomatous leaf cuticular membranes of Hedera helix L. was measured for uncharged hydrophilic (octanol/water partition coefficient log K(O/W) < or =0) and lipophilic compounds (log K(O/W) >0). The set of compounds included lipophilic plant protection agents, hydrophilic carbohydrates, and the volatile compounds water and ethanol. Plotting the mobility of the model compounds versus the molar volume resulted in a clear differentiation between a lipophilic and a hydrophilic pathway. The size selectivity of the lipophilic pathway was described by the free volume theory. The pronounced tortuosity of the diffusional path was caused by cuticular waxes, leading to an increase in permeance for the lipophilic compounds after wax extraction. The size selectivity of the hydrophilic pathway was described by hindered diffusion in narrow pores of molecular dimensions. A distinct increase in size selectivity was observed for hydrophilic compounds with a molar volume higher than 110 cm3 mol(-1). Correspondingly, the size distribution of passable hydrophilic pathways was interpreted as a normal distribution with a mean pore radius of 0.3 nm and a standard deviation of 0.02 nm. The increased permeance of the hydrophilic compounds by the removal of cuticular waxes was attributed to an increase in the porosity, a decrease in the tortuosity, and a widening of the pore size distribution. Cuticular transpiration resulted from the permeation of water across the hydrophilic pathway. The far-reaching implications of two parallel pathways for the establishment of correlations between cuticular structure, chemistry, and function are discussed.     

13C NMR studies of D- and L-phenylalanine binding to cobalt(II) carboxypeptidase A

13C NMR T1 and T2 measurements have been performed on cobalt(II) substituted carboxypeptidase A in the presence of carboxylate-13C-enriched L- and D-phenylalanine. Upon binding to the cobalt enzyme, the longitudinal and transverse relaxation rates T1p-1 and T2p-1 of these inhibitors are enhanced significantly compared to the zinc enzyme, allowing both determination of an affinity constant for inhibitor binding, K, and calculation of the metal-13C carboxylate distances. The L-and D- Phe concentration dependence of T2p-1 yields affinity constants of 290 +/- 60M-1 and 670 +/- 90M-1. The distance measurements calculated for Co-13C from T1p-1 are 0.39 +/- 0.04 and 0.42 +/- 0.04 nm for L-Phe and D-Phe. Both values are too great for direct coordination of their carboxylate groups to the metal atom. Upon formation of their respective ternary enzyme.Phe.N3- complexes, the distances are essentially unaltered. In conjunction with electronic absorption studies on these complexes it can be concluded that N3-, but not the amino acid carboxylate, is bound to the metal.