2H and 13C nuclear magnetic resonance study of N-palmitoylgalactosylsphingosine (cerebroside)/cholesterol bilayers.

13C- and 2H-NMR experiments were used to examine the phase behavior and dynamic structures of N-palmitoylgalactosylsphingosine (NPGS) (cerebroside) and cholesterol (CHOL) in binary mixtures. 13C spectra of 13C=O-labeled and 2H spectra of [7,7-2H2] chain-labeled NPGS as well as 3 alpha-2H1 CHOL indicate that cerebroside and CHOL are immiscible in binary mixtures at temperatures less than 40 degrees C. In contrast, at 40 degrees C < t < or = T(C) (NPGS), up to 50 mol% CHOL can be incorporated into melted cerebroside bilayers. In addition, 13C and 2H spectra of melted NPGS/CHOL bilayers show a temperature and cholesterol concentration dependence. An analysis of spectra obtained from the melted 13C=O NPGS bilayer phase suggests that the planar NH-C=O group assumes an orientation tilted 40 degrees-55 degrees down from the bilayer interface. The similarity between the orientation of the amide group relative to the bilayer interface in melted bilayers and in the crystal structure of cerebroside suggests that the overall crystallographic conformation of cerebroside is preserved to a large degree in hydrated bilayers. Variation of temperature from 73 degrees to 86 degrees C and CHOL concentration from 0 to 51 mol% results in small changes in this general orientation of the amide group. 2H spectra of chain-labeled NPGS and labeled CHOL in NPGS/CHOL bilayer demonstrate that molecular exchange between the gel and liquid-gel (LG) phases is slow on the 2H time scale, and this facilitates the simulation of the two component 2H spectra of [7,7-2H2]NPGS/CHOL mixtures. Simulation parameters are used to quantitate the fractions of gel and LG cerebroside. The quadrupole splitting of [7,7-2H2]NPGS/CHOL mixtures and 2H simulations allows the LG phase bilayer fraction to be characterized as an equimolar mixture of cerebroside and CHOL.     

Altered hydrogen bonding of Arg82 during the proton pump cycle of bacteriorhodopsin: a low-temperature polarized FTIR spectroscopic study

Light-driven proton transport in bacteriorhodopsin (BR) is achieved by dynamic rearrangement of the hydrogen-bonding network inside the membrane protein. Arg82 is located between the Schiff base region and proton release group, and has a major influence on the pK(a) values of these groups. It is believed that Arg82 changes its hydrogen-bonding acceptors during the pump cycle of BR, stages of which are correlated with proton movement along the transport pathway. In this study, we compare low-temperature polarized FTIR spectra of [eta(1,2)-(15)N]arginine-labeled BR in the 2750-2000 cm(-1) region with those of unlabeled BR for the K, L, M, and N intermediates. In the K-minus-BR difference spectra, (15)N-shifted modes were found at 2292 (-)/2266 (+) cm(-1) and at 2579 (-)/2567 (+) cm(-1). The former corresponds to strong hydrogen bonding, while the latter corresponds to very weak hydrogen bonding. Both N-D stretches probably originate from Arg82, the former oriented toward water 406 and the latter toward the extracellular side, and both hydrogen bonds are somewhat strengthened upon retinal photoisomerization. This perturbation of arginine hydrogen bonding is entirely relaxed in the L intermediate where no (15)N-isotope shifts are observed in the difference spectrum. In the M intermediate, the frequency is not significantly altered from that in BR. However, the polarized FTIR spectra strongly suggest that the dipolar orientation of the strongly hydrogen bonded N-D group of Arg82 is changed from perpendicular to parallel to the membrane plane. Such a change is presumably related to the motion of the Arg82 side chain from the Schiff base region to the extracellular proton release group. Additional bands corresponding to weak hydrogen bonding were observed in both the M-minus-BR and N-minus-BR spectra. Changes in hydrogen-bonding structures involving Arg82 are discussed on the basis of these FTIR observations.     

Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes

Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, coupled to lissamine-rhodamine, fluoresceine, eosine and NBD. The spectroscopic properties of these compounds, in different solvent systems and when incorporated into micelles of a non-ionic detergent or liposomes of a phospholipid, are reported. Incubation of these respective sulfatides with a human leukocyte preparation, resulted in the formation of the corresponding fluorescent cerebrosides.     

The effect of hydrogen bonds on the conformation of glycosphingolipids. Methylated and unmethylated cerebroside studied by X-ray single crystal analysis and model calculations

The conformation and molecular packing of permethylated beta-D-galactosyl-N-octadecanoyl-D-spingosine (cerebroside) was determined by X-ray single crystal analysis at 185 K (R = 0.16). The lipid crystallizes in the orthorhombic space group P2(1)2(1)2(1) with the unit cell dimensions a = 8.03, b = 7.04 and c = 88.10 A. The four molecules in the unit cell pack in a bilayer arrangement with tilting (48 degrees) hydrocarbon chains. The direction of the chain tilt alternates in the two bilayer halves and in adjacent bilayers. In order to define the effect of hydrogen bonds on the molecular conformation the structural features of the permethylated cerebroside are compared with that of unsubstituted cerebroside (I. Pascher and S. Sundell (1977) Chem. Phys. Lipids 20, 179). It is shown that methylation of the hydrogen donor groups does not affect the conformation of the ceramide part. However, by abolishing the intramolecular hydrogen bond between the amide N--H group and the glycosidic oxygen the galactose ring changes its orientation from layer-parallel to layer-perpendicular. Calculations using molecular mechanics, MM2(87), show that in natural cerebroside the intramolecular hydrogen bond stabilizes the theta 1 = -syn-clinal conformation about the C(1)--C(2) sphingosine bond by 2-2.5 kcal/mol compared to other staggered conformations. The significance of the L shape of the native cerebroside, making both the carbohydrate and polar ceramide groups accessible as a binding epitope in recognition processes, is discussed.     

Synthesis and characterization of MMA–NaAlg/hydroxyapatite composite and the interface analyse with molecular dynamics

A novel composite of α-methyl methacrylate (MMA) grafted sodium alginate (NaAlg) and hydroxyapatite (HA) was prepared in this study. The compositions and chemical groups of materials were investigated by infrared spectra (IR), X-ray diffraction (XRD) and nuclear magnetic resonance (NMR). The results showed that MMA has been successfully grafted with the hydroxyl group of sodium alginate. Moreover many chemical bonds existed in the composite, including the “egg-box” structure and hydrogen bonding. Meanwhile, the chemical bondings between MMA and sodium alginate partly replaced the intermolecular hydrogen bonding or the intramolecular hydrogen bonding in sodium alginate. The composite had better water contact angle than sodium alginate, indicating the strong hydrophilic character of pure sodium alginate was improved. The molecular dynamics (MD) method was used to simulate and evaluate the interaction energies based on the theoretics, which suggested that the copolymer whose every monomer grafted with one MMA had a more stable structure.     

Differential scanning calorimetric and Fourier transform infrared spectroscopic investigations of cerebroside polymorphism

Calorimetric and Fourier transform infrared (FTIR) spectroscopic studies have been made of the polymorphism exhibited by bovine brain cerebroside-water systems, and the effect of cholesterol and dipalmitoylphosphatidylcholine (DPPC) upon this polymorphism was investigated. The conversion of the cerebroside from the thermodynamically stable to the metastable form is found to be accompanied by spectral changes, indicating a decrease in cerebroside headgroup hydration and a rearrangement of the hydrogen-bond network. The incorporation of low concentrations of cholesterol and DPPC into cerebroside bilayers broadens the thermal transitions associated with the cerebroside as a result of the disruption of cerebroside-cerebroside interactions. This disruption is evident in the spectra of cerebroside/cholesterol mixtures.     

Electronic spectroscopy and solvatochromism in the chromophore of GFP and the Y66F mutant.

The electronic spectra of the chromophore of the wild type green fluorescent protein, GFP, and of a mutant form Y66F GFP in which the chromophore lacks the hydroxyl group have been studied. The acid-base properties, solvatochromism, vibronic structure and edge excitation red shift have all been measured. The results are compared with the spectra of the chromophore in the protein environment. These data suggest that the transition energy for the GFP chromophore is influenced by a number of factors in its environment, and in particular by hydrogen bonding.     

On the effect of temperature on the ultraviolet spectra of protein chromophores

The thermal perturbation difference spectra - TPDS (15-30 degrees C) - of N-acetyl-tyrosine-ethyl ester and o-methyl-N-acetyl-tyrosine were studied in ethyl acetate and dimethyl ether with/without the addition of butanol which served as a proton donor in hydrogen bonding. In all cases the longwave shift of the absorption spectrum is shown to be a principal factor that determines the origin of TPDS and the hydrogen bonding has no effect on these spectra. These results contradict the view that the red shift of protein chromophore spectra at the elevation of temperature is a unique feature of water as a solvent. The water-inaccessible chromophores in proteins may be perturbed by temperature increase, producing red shift.     

Effect of ethylene glycol on the phase transition kinetics of gluco- and galactocerebrosides

The effect of different concentrations of ethylene glycol in water on the phase transition (metastable----stable state) of Gaucher's glucocerebroside, of bovine brain cerebroside type II (non hydroxy acyl chains only) and of N-palmitoylgalactocerebroside has been investigated. The phase transition and its kinetics were inferred from the thermograms at different heating and cooling rates and confirmed by FTIR spectra of the cerebrosides in the different states. The significance of the conformational differences of the glucose and of the galactose residues with respect to their solvation, and the subsequent effect on the intermolecular interactions and the phase transition is discussed.     

Conformational studies of alanine oligopeptides by nuclear magnetic resonance.

We have studied the nmr spectra of the series of alanine oligopeptides containing a methoxyethoxyethoxyacetyl blocking group on the N-terminal residue and a morpholino blocking group on the C-terminal residue. Spectra were measured in chloroform–trifluoroacetic acid solvent systems. For oligomers with chain lengths of five or more, “double peaks” are observed for the α-CH protons. Addition of trifluoroacetic acid causes the peaks to coalesce. The amount of trifluoroacetic acid necessary for coalescence increases from the pentamer to the nonamer. These findings are general since alanine oligomers with different blocking groups exhibit similar “double peak” phenomena. We explain the “double peak” phenomenon in terms of specific folded forms of the oligopeptides which arise from intramolecular hydrogen bonding. Additional evidence for such hydrogen bonding is presented based upon infrared studies. Slight aggregation probably occurs for the pentamer and hexamer which may stabilize the folded forms.     

Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol

The ESR spectra from different positional isomers of sphingomyelin and phosphatidylcholine spin-labeled in their acyl chain have been studied in sphingomyelin(cerebroside)-phosphatidylcholine mixed membranes that contain cholesterol. The aim was to investigate mechanisms by which cholesterol could stabilize possible domain formation in sphingolipid-glycerolipid membranes. The outer hyperfine splittings in the ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled on the 5 C atom of the acyl chain were consistent with mixing of the components, but the perturbations on adding cholesterol were greater in the membranes containing sphingomyelin than in those containing phosphatidylcholine. Infrared spectra of the amide I band of egg sphingomyelin were shifted and broadened in the presence of cholesterol to a greater extent than the carbonyl band of phosphatidylcholine, which was affected very little by cholesterol. Two-component ESR spectra were observed from lipids spin-labeled on the 14 C atom of the acyl chain in cholesterol-containing membranes composed of sphingolipids, with or without glycerolipids (sphingomyelin/cerebroside and sphingomyelin/cerebroside/phosphatidylcholine mixtures). These results indicate the existence of gel-phase domains in otherwise liquid-ordered membranes that contain cholesterol. In the gel phase of egg sphingomyelin, the outer hyperfine splittings of sphingomyelin spin-labeled on the 14-C atom of the acyl chain are smaller than those for the corresponding spin-labeled phosphatidylcholine. In the presence of cholesterol, this situation is reversed; the outer splitting of 14-C spin-labeled sphingomyelin is then greater than that of 14-C spin-labeled phosphatidylcholine. This result provides some support for the suggestion that transbilayer interdigitation induced by cholesterol stabilizes the coexistence of gel-phase and "liquid-ordered" domains in membranes containing sphingolipids.     

Interaction of myelin basic protein with different ionization states of phosphatidic acid and phosphatidylserine

Myelin basic protein (BP) has a perturbing effect on some lipids, causing, among other effects, a decrease in the temperature and enthalpy of the phase transition. This is believed to be a result of penetration of some hydrophobic residues of the protein partway into the lipid bilayer. Variations in the perturbing effect of BP on different acidic lipids has been attributed to the ability of the lipids to participate in intermolecular hydrogen bonding which inhibits penetration of the protein. Participation in intermolecular hydrogen bonding depends on the ionization state of the lipid as well as the type of lipid. In order to further test the dependence of the degree of penetration of BP on the intermolecular hydrogen bonding properties of lipids, the effect of BP on the phase transition of lipids in different ionization states was studied using differential scanning calorimetry. Dipalmitoylphosphatidic acid (DPPA) and dimyristoylphosphatidylserine (DMPS) were studied at different pH-values from 4 to 9.5. The results were compared to data obtained earlier with phosphatidylglycerol (PG), which is in the same ionization state at pH-values above 4, in order to distinguish the effects of pH on the protein from effects on the lipids. The perturbing effect of BP on PG increases with increase in pH. This is probably a result of the increasing hydrophobicity of the protein as the histidines become deprotonated, which allows greater penetration of the protein into the bilayer. In contrast, the effect on DPPA was greatest at low pH, where the state of ionization of the lipid is less than 1 and protein binding utilizes all of the hydrogen bond accepting sites (P-O-) on the lipid. BP had no perturbing effect on DPPA at higher pH where the state of ionization is between 1 and 1.5, and hydrogen bond accepting and donating sites (P-OH) are still available even after binding of the protein. Thus hydrogen bonding occurs at high pH and penetration of hydrophobic residues of the protein into DPPA is inhibited. BP had a large perturbing effect on DMPS at all pH values above 4 suggesting that lipid intermolecular hydrogen bonding does not occur in the presence of the protein and its hydrophobic residues consequently can penetrate into the bilayer. The protein may inhibit hydrogen bonding by binding electrostatically to the anionic hydrogen bond accepting group of PS.     

Vibrational (FT-IR and FT-Raman) spectral investigations of 7-aminoflavone with density functional theoretical simulations

FT-Raman and FT-IR spectra of the 7-aminoflavone have been recorded and analysed. The detailed interpretation of the vibrational spectra has been carried out with the aid of normal coordinate analysis following the scaled quantum mechanical force field methodology. The various intramolecular interactions that are responsible for stabilisation of the molecule were revealed by natural bond orbital analysis. The obtained vibrational wavenumbers and optimised geometric parameters were observed to be in good agreement with the experimental data. The carbonyl stretching vibrations have been lowered due to conjugation and hydrogen bonding in the molecules.     

Solid state NMR studies of hydrogen bonding in a citrate synthase inhibitor complex.

The ionization state and hydrogen bonding environment of the transition state analogue (TSA) inhibitor, carboxymethyldethia coenzyme A (CMX), bound to citrate synthase have been investigated using solid state NMR. This enzyme-inhibitor complex has been studied in connection with the postulated contribution of short hydrogen bonds to binding energies and enzyme catalysis: the X-ray crystal structure of this complex revealed an unusually short hydrogen bond between the carboxylate group of the inhibitor and an aspartic acid side chain [Usher et al. (1994) Biochemistry 33, 7753-7759]. To further investigate the nature of this short hydrogen bond, low spinning speed 13C NMR spectra of the CMX-citrate synthase complex were obtained under a variety of sample conditions. Tensor values describing the chemical shift anisotropy of the carboxyl groups of the inhibitor were obtained by simulating MAS spectra (233 +/- 4, 206 +/- 5, and 105 +/- 2 ppm vs TMS). Comparison of these values with our previously reported database and ab initio calculations of carbon shift tensor values clearly indicates that the carboxyl is deprotonated. New data from model compounds suggest that hydrogen bonds in a syn arrangement with respect to the carboxylate group have a pronounced effect upon the shift tensors for the carboxylate, while anti hydrogen bonds, regardless of their length, apparently do not perturb the shift tensors of the carboxyl group. Thus the tensor values for the enzyme-inhibitor complex could be consistent with either a very long syn hydrogen bond or an anti hydrogen bond; the latter would agree very well with previous crystallographic results. Two-dimensional 1H-13C heteronuclear correlation spectra of the enzyme-inhibitor complex were obtained. Strong cross-peaks were observed from the carboxyl carbon to proton(s) with chemical shift(s) of 22 +/- 5 ppm. Both the proton chemical shift and the intensity of the cross-peak indicate a very short hydrogen bond to the carboxyl group of the inhibitor, the C.H distance based upon the cross-peak intensity being 2.0 +/- 0.4 A. This proton resonance is assigned to Hdelta2 of Asp 375, on the basis of comparison with crystal structures and the fact that this cross-peak was absent in the heteronuclear correlation spectrum of the inhibitor-D375G mutant enzyme complex. In summary, our NMR studies support the suggestion that a very short hydrogen bond is formed between the TSA and the Asp carboxylate.     

Role of threonine 101 on the stability of the heme active site of cytochrome P450cam: multiwavelength circular dichroism studies

The role of the threonine 101 residue that resides close to the heme propionic acid side chain of cytochrome P450cam on the conformational properties of the active site of the enzyme has been investigated by circular dichroism (CD) spectroscopy. Site-specific mutation of the threonine by valine has been carried out that does not affect the size of the residue but significantly alters the hydropathy index. The T101V mutant of cytochrome P450cam showed distinct differences in the CD spectra near the heme region, indicating a subtle effect of the mutation on the properties of the heme active site. Thermal stabilities of the mutant and wild-type enzyme have been studied by temperature dependence of the ellipticity (intensity of the CD band) in the far-UV region for the secondary structure and at different wavelengths in the visible region that arise from the heme moiety for the tertiary structure around the prosthetic group. The thermal unfolding data from variations of the CD intensity at different wavelengths were analyzed using a generalized multistep unfolding model, and two distinct equilibrium intermediate conformational states of the enzyme were identified. The mutation of the T101 residue by valine was found to decrease the thermal stability of both the intermediates in the presence of the substrate. On the other hand, this mutation had no apparent effect on the thermal stability of the enzyme in the absence of the substrate. These results suggested that the threonine residue stabilizes the protein cavity around the heme center in the case of the substrate-bound species, possibly by hydrogen bonding with one of the propionate side chains of the heme moiety. Such hydrogen bonding of the heme propionate with threonine is absent in the substrate-free form of the enzyme.     

Computational study of the absorption spectra of green fluorescent protein mutants

In this work, we present a theoretical study of the relationship between molecular structure and the red-shift in absorption spectra of S65G and S65T green fluorescent protein (GFP) mutants. To identify the effects of the protein environment, we combined results from molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics calculations to obtain structural properties, and applied time-dependent density functional theory to calculate the excitation energies. By using results from the MD simulations, we were able to provide a systematic analysis of the structural details that may effect the red-shift in the absorption spectra when taking into account temperature effects. Furthermore, a detailed study of hydrogen bonding during the MD simulations demonstrated differences between S65G and S65T, for example, regarding hydrogen bonding with Glu222. An analysis of the absorption spectra for different forms of the chromophore emphasized the dominance of the anionic forms in solution for the S65G and S65T GFP mutants.     

Equilenin: a specific fluorescent probe for steroid—protein interactions in sex steroid-binding protein

Equilenin, a naturally fluorescent steroid, has high binding affinity for human sex steroid-binding protein (SBP). At 4°C the equilibrium association constant is ～6 × 10⁷ M^?1. The fluorescence excitation and emission spectra of the steroid—protein complex indicate that both hydrophobic interactions and hydrogen bonding of the 3'-hydroxyl group of the estrogen are important in its binding to the protein. Equilenin has a substantially different 3-dimensional spatial configuration compared with the normally bound androgens, and yet exhibits very tight binding to SBP. This suggests that SBP undergoes a conformational change to accomodate equilenin.     

Effect of fatty acid chain length,fatty acid hydroxylation,and various cations on phase behavior of synthetic cerebroside sulfate

Synthetic species of CBS containing palmitic, stearic, lignoceric, D-2-hydroxy palmitic, or D-2-hydroxy stearic acid were prepared and their phase behavior in the presence of a number of mono- and divalent cations was studied by differential scanning calorimetry and the use of fatty acid spin labels. The results showed that both the non-hydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) forms of cerebroside sulfate (CBS) can occur in two different gel states, a metastable state and a lower entropy stable state. The phase behavior is more sensitive to the type and concentration of cation present than in the case with acidic phospholipids. The sensitivity of the transition temperature (T_m) to cation concentration reflects, in part, increased participation of the lipid in intermolecular hydrogen bonding interactions as the negative charge of the sulfate is shielded. The extra hydroxyl group on the HFA also contributes to the intermolecular hydrogen bonding network causing a significant increase in the T_m.The HFA has an even more significant effect in causing inhibition of formation of the stable state. Formation of the stable state is also inhibited by Li⁺ and divalent cations. A similar mechanism may be involved, i.e.; cross-linking of adjacent lipids or increased intermolecular interactions inhibit the molecular rearrangement necessary to form the stable state. This inhibition is counteracted by an increase in fatty acid chain length. The results suggest that the stable state may be interdigitated as a result of the unequal chain length between the sphingosine base and the fatty acid.