Structure of DNA hydration shells studied by Raman spectroscopy

We have used Raman scattering to study the water O-H stretching modes at approximately 3450 and approximately 3220 cm-1 in DNA films as a function of relative humidity (r.h.). The intensity of the 3220-cm-1 band vanishes as the r.h. is decreased from 98% to around 80%, which indicates that the hydrogen-bond network of water is disrupted in the primary hydration shell (which therefore cannot have an "ice-like" structure). The number of water molecules in the primary hydration shell was determined from the intensity of the approximately 3200-cm-1 band as about 30 water molecules per nucleotide pair. The approximately 3400-cm-1 O-H stretch band was used for determining the total water content, and this band persists at 0% r.h., implying that 5-6 tightly bound water molecules per nucleotide pair remain. The frequency of the approximately 3400-cm-1 O-H stretch mode is lower by 30 to 45 cm-1 in the primary hydration shell compared to free water. The water content as a function of r.h. obtained from these experiments agrees with gravimetric measurements. The disappearance of the approximately 3200-cm-1 band and the shift of the approximately 3400-cm-1 O-H stretch band provide a reliable way of measuring the hydration number of DNA.     

Orientation of specifically 13C=O labeled phosphatidylcholine multilayers from polarized attenuated total reflection FT-IR spectroscopy.

Oriented multilayers of 1-myristoyl-2(1-13C)-myristoyl-sn-glycero-3-phosphatidylcholine (2[1-13C]DMPC) and 1-palmitoyl-2(1-13C)-palmitoyl-sn-glycero-3-phosphatidylcholine (2[1-13C]DPPC) were investigated by use of attenuated total reflection infrared spectroscopy with polarized light. Experiments were performed with the aim to determine the orientation of the two ester groups in these phospholipids in the solid state and in the hydrated state at temperatures below and above the respective gel to liquid-crystalline phase transitions. Substitution of the naturally occurring 12C carbonyl carbon atom by 13C in the ester group of the sn-2 chain of DMPC and DPPC shifts the infrared absorption of the carbonyl double bond stretching vibration to lower frequency. This results in two well-resolved ester C=O bands which can be assigned unequivocally to the sn-1 and sn-2 chains as they are separated by more than 40 cm-1. The two ester CO-O single bond stretching vibrations of the molecular fragments-CH2CO-OC-are also affected and the corresponding infrared absorption band shifts by 20 cm-1 on 13C-labeling of the carbonyl carbon atom. From the dichroic ratios of the individual ester bands in 2(1-13C)DMPC and 2(1-13C)DPPC we were able to demonstrate that the sn-1 and sn-2 ester C=O groups are similarly oriented with respect to the bilayer plane, with an angle greater than or equal to 60 degrees relative to the bilayer normal. The two CO-O single bonds on the other hand have very different orientations. The CH2CO-OC fragment of the sn-1 chain is oriented along the direction of the all-trans methylene chain, whereas the same molecular segment of the sn-2 carbon chain is directed toward the bilayer plane. This orientation of the ester groups is retained in the liquid-crystalline phase. The tilt angle of the hydrocarbon all-trans chains, relative to the membrane normal, is 25 degrees in the solid state of DMPC and DPPC multibilayers. In the hydrated gel state this angle varies between 26 degrees and 30 degrees, depending on temperature. Neither the orientation of the phosphate group, nor that of the choline group varies significantly in the different physical states of these phospholipids.     

Interaction of ferricytochrome c with zwitterionic phospholipid bilayers: a Raman spectroscopic study

The vibrational Raman spectra of both pure L-alpha-dipalmitoylphosphatidylcholine (DPPC) liposomes and DPPC multilayers reconstituted with ferricytochrome c under varying conditions of pH and ionic strength are reported as a function of temperature. Total integrated band intensities and relative peak height intensity ratios, two spectral scattering parameters used to determine bilayer disorder, are invariant to changes in pH and ionic strength but exhibit a sensitivity to the bilayer concentration of the ferricytochrome c. Protein concentrations were estimated by comparing the 1636 cm-1 resonance Raman line of known ferricytochrome c solutions to intensity values for the reconstituted multilayer samples. Temperature-dependent profiles of the 3100-2800 cm-1 C-H stretching, 1150-1000 cm-1 C-C stretching, 1440 cm-1 CH2 deformation, and 1295 cm-1 CH2 twisting mode regions characteristic of acyl chain vibrations reflect bilayer perturbations due to the weak interactions of ferricytochrome c. The DPPC multilamellar gel to liquid-crystalline phase transition temperature, TM, defined by either the C-H stretching mode I2935/I2880 or the C-C stretching mode I1061/I1090 peak height intensity ratios, is decreased by approximately 4 degrees C for the approximately 10(-4) M ferricytochrome c reconstituted DPPC liposomes. Other spectral features, such as the increase in the 2935 cm-1 C-H stretching mode region and the enhancement of higher frequency CH2 twisting modes, which arise in bilayers containing approximately 10(-4) M protein, are interpreted in terms of protein penetration into the hydrophobic region of the bilayer.     

Water structural changes in the L and M photocycle intermediates of bacteriorhodopsin as revealed by time-resolved step-scan Fourier transform infrared (FTIR) spectroscopy

In previous Fourier transform infrared (FTIR) studies of the photocycle intermediates of bacteriorhodopsin at cryogenic temperatures, water molecules were observed in the L intermediate, in the region surrounded by protein residues between the Schiff base and Asp96. In the M intermediate, the water molecules had moved away toward the Phe219-Thr46 region. To evaluate the relevance of this scheme at room temperature, time-resolved FTIR difference spectra of bacteriorhodopsin, including the water O-H stretching vibration frequency regions, were recorded in the micro- and millisecond time ranges. Vibrational changes of weakly hydrogen-bonded water molecules were observed in L, M, and N. In each of these intermediates, the depletion of a water O-H stretching vibration at 3645 cm-1, originating from the initial unphotolyzed bacteriorhodopsin, was observed as a trough in the difference spectrum. This vibration is due to the dangling O-H group of a water molecule, which interacts with Asp85, and its absence in each of these intermediates indicates that there is perturbation of this O-H group. The formation of M is accompanied by the appearance of water O-H stretching vibrations at 3670 and 3657 cm-1, the latter of which persists to N. The 3670 cm-1 band of M is due to water molecules present in the region surrounded by Thr46, Asp96, and Phe219. The formation of L at 298 K is accompanied by the perturbations of Asp96 and the Schiff base, although in different ways from what is observed at 170 K. Changes in a broad water vibrational feature, centered around 3610 cm-1, are kinetically correlated with the L-M transition. These results imply that, even at room temperature, water molecules interact with Asp96 and the Schiff base in L, although with a less rigid structure than at cryogenic temperatures.     

Interaction of 7,7,8,8-tetracyanoquinodimethane with diacylphosphatidylcholines and -phosphatidylglycerols. A photoacoustic Fourier transform infrared study

7,7,8,8-Tetracyanoquinodimethane (TCNQ) was incorporated in fully hydrated liposomes of the following pyrene-containing as well as non-labelled phospholipids: 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatid ylc holine (PPDPC), 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidyl- rac'- glycerol (rac'-PPDPG), 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidyl- sn-3'- glycerol (3'-PPDPG), 1-[10-(pyren-1-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl- sn-3'- glycerol (3'-PDPPG), 1-[10-pyren-1-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl-s n-1'- glycerol (1'-PDPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl-rac'-glycerol (rac'-DPPG). Lyophilized charge-transfer (CT) complexes of TCNQ with phospholipids were examined by Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS). Due to the spectral changes observed in the vibrational bands originating from the CH2 and C = O stretching vibrations, and the bands associated with the polar headgroup of the phospholipids it is evident that TCNQ has only a minor perturbing effect on the hydrocarbon chains. However, the molecular interaction between TCNQ and phospholipids is seen in the polar headgroup region. The donated electrons are most likely located on the oxygens of the phosphate group in the polar head. As judged from the present infrared data interactions of TCNQ with phosphatidylcholines (PC) and phosphatidylglycerols (PG) differ. For PG the complex formation produces a second strong C = O stretching band at approx. 1710 cm-1 in addition to the band at approx. 1735 cm-1 indicating a specific molecular interaction in the interfacial region.     

A resonance Raman study on a reaction intermediate of Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating).

Resonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i.e., [1-13C], [2-13C], [ring-U-13C6], and [15N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of D-amino acid oxidase (DAO). The isotope effects on the 1,665 cm-1 band with [15N] or [2-13C]phenylalanine indicate that the band is due to the C = N stretching mode of an imino acid derived from phenylalanine, i.e., alpha-imino-beta-phenylpropionate. The intense band at 1,389 cm-1 is contributed to by the CO2- symmetric stretching and C-CO2- stretching modes of alpha-imino-beta-phenylpropionate. The 1,602 cm-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1,605 cm-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10a) = C(4a) - C(4) = O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a) = C(4a) - C(4) = O of reduced flavin and the plane containing H2+N = C - CO2- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.     

Hydrogen bonding between anhydrous cholesterol and phosphatidylcholines: an infrared spectroscopic study

Fourier transform infrared spectroscopy performed with a high pressure diamond anvil cell was used to study hydrogen bonding between anhydrous phosphatidylcholines and cholesterol at the molar ratio 4:1. The hydroxyl group of cholesterol which acts as a proton donor, engages in strong hydrogen bonding to the sn-2 chain carbonyl group of DMPC, DPPC and HPPC and in weak hydrogen bonding to the phosphate group of all these phospholipids. No evidence of hydrogen bonding between cholesterol and the sn-1 chain carbonyl group of DMPC and DPPC was found. From a comparison of the relative hydrogen-bond strengths between cholesterol or water and the sn-2 chain carbonyl and phosphate groups of all these phospholipids, it is predicted that in aqueous dispersions of cholesterol containing phospholipids, the hydrogen bond of cholesterol to the phosphate group would be replaced by that of water, while the hydrogen bond of cholesterol to the sn-2 chain carbonyl group would remain intact.     

ATP-Induced phosphorylation of the sarcoplasmic reticulum Ca2+ ATPase: molecular interpretation of infrared difference spectra.

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and from [gamma-18O3]caged ATP. A band at 1546 cm-1, not observed with the deuterated enzyme, can be assigned to the amide II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP binding. This is also indicated between 1700 and 1610 cm-1, where bandshifts of up to 10 cm-1 observed upon protein deuteration suggest that amide I modes of the protein backbone dominate the difference spectrum. From the band positions it is deduced that alpha-helical, beta-sheet, and probably beta-turn structures are affected in the phosphorylation reaction. Model spectra of acetyl phosphate, acetate, ATP, and ADP suggest the tentative assignment of some of the bands of the phosphorylation spectrum to the molecular groups of ATP and Asp351, which participate directly in the phosphate transfer reaction: a positive band at 1719 cm-1 to the C==O group of aspartyl phosphate, a negative band at 1239 cm-1 to the nuas(PO2-) modes of the bound ATP molecule, and a positive band at 1131 cm-1 to the nuas(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported by the band's sensitivity toward isotopic substitution in the gamma-phosphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become partly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C==O group of aspartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gln, and Arg residues was not detected.     

Characterization of Langmuir-Blodgett films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphat idylcholine by FTIR-ATR

Monomolecular films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidylc holine (PPDPC) were transferred from an air/water interface onto a germanium attenuated total reflection crystal by the Langmuir-Blodgett (LB) technique. The assemblies were thereafter investigated by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. To determine the molecular organization in the deposited layers we monitored the CH2 and C = O stretching and the CH2 bending regions of the infrared spectra of these lipids in detail. Using Fourier self-deconvolution technique, the carbonyl stretching mode was resolved into two models corresponding to the conformational differences in the ester linkages of the phospholipid sn-1 and sn-2 acyl chains. By varying the temperature of the subphase and using different surface pressures, we were able to transfer different conformational states of DPPC onto a germanium ATR crystal. Deposition of DPPC at 40 mN m-1 and at 15 degrees C or at 20 mN m-1 and at 35 degrees C results in LB-assemblies in ordered or disordered states, respectively, as judged by the IR spectra. These structures in LB films correspond to the state of DPPC in liposomes below and above the temperature of the order-disorder phase transition. Irrespective of the surface pressure and subphase temperature used during the deposition, an ordering process was found in DPPC films when the number of the transferred layers was increased from one to five. The pyrene-labelled phosphatidylcholine analogue, PPDPC, behaved differently from DPPC. In the case where one to three layers of PPDPC transferred at 35 mN m-1 and at 20 degrees C only conformational structures resembling those in fully hydrated liposomes above the main transition temperature were observed.     

Electrophilic catalysis in triosephosphate isomerase: the role of histidine-95

Electrophilic catalysis by histidine-95 in triosephosphate isomerase has been probed by using Fourier transform infrared spectroscopy and X-ray crystallography. The carbonyl stretching frequency of dihydroxyacetone phosphate bound to the wild-type enzyme is known to be 19 cm-1 lower (at 1713 cm-1) than that of dihydroxyacetone phosphate free in solution (at 1732 cm-1), and this decrease in stretching frequency has been ascribed to an enzymic electrophile that polarizes the substrate carbonyl group toward the transition state for the enolization. Infrared spectra of substrate bound to two site-directed mutants of yeast triosephosphate isomerase in which histidine-95 has been changed to glutamine or to asparagine show unperturbed carbonyl stretching frequencies between 1732 and 1742 cm-1. The lack of carbonyl polarization when histidine-95 is removed suggests that histidine-95 is indeed the catalytic electrophile, at least for dihydroxyacetone phosphate. Kinetic studies of the glutamine mutant (H95Q) have shown that the enzyme follows a subtly different mechanism of proton transfers involving only a single acid-base catalytic group. These findings suggest an additional role for histidine-95 as a general acid-base catalyst in the wild-type enzyme. The X-ray crystal structure of the H95Q mutant with an intermediate analogue, phosphoglycolohydroxamate, bound at the active site has been solved to 2.8-A resolution, and this structure clearly implicates glutamate-165, the catalytic base in the wild-type isomerase, as the sole acid-base catalyst for the mutant enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization and orientation of functional water molecules in bacteriorhodopsin as revealed by polarized Fourier transform infrared spectroscopy.

Linear dichroic difference Fourier transform infrared spectra upon formation of the M photointermediate were recorded with oriented purple membranes. The purpose was to determine the angle of the directions of the dipole moments of 1) the water molecule whose O-H stretching vibration appears at 3643 cm-1 for the unphotolyzed state and 3671 cm-1 for the M intermediate, and 2) the C=O bond of protonated Asp85 in the M intermediate. The angle of 36 degrees we find for the C=O of the protonated Asp85 in the M intermediate is not markedly different from 26 degrees for unprotonated Asp85 in the model based on cryoelectron diffraction, indicating the absence of gross orientation changes in Asp85 upon its protonation. The O-H band at 3671 cm-1 of a water molecule in the M intermediate, although its position has not determined, is fixed almost parallel to the membrane plane. For the unphotolyzed state the angle of the water O-H to the membrane normal was determined to be 60 degrees. On the basis of these data and the structural model, we place the water molecule in the unphotolyzed state at a position where it forms hydrogen bonds with the Schiff base, Asp85, Asp212, and Trp86.     

Unusually strong H-bonding to the heme ligand and fast geminate recombination dynamics of the carbon monoxide complex of Bacillus subtilis truncated hemoglobin

The active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at 545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were observed in infrared absorption and resonance Raman measurements. The very low C-O stretching frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1) indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogen-bonding network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489 cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis. Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization.     

Water structural changes in the bacteriorhodopsin photocycle: analysis by Fourier transform infrared spectroscopy.

The Fourier transform infrared difference spectra between light-adapted bacteriorhodopsin (BR) and its photointermediates, L and M, were analyzed for the 3750-3450-cm-1 region. The O-H stretching vibrational bands were identified from spectra upon substitution with 2H2O. Among them, the 3642-cm-1 band of BR was assigned to water by substitution with H2(18)O. By a comparison with the published infrared spectra of the water in model systems [Mohr, S.C., Wilk, W.D., & Barrow, G.M. (1965) J. Am. Chem. Soc. 87, 3048-3052], it is shown that the O-H bonds of the water in BR interact very weakly. Upon formation of L, the interaction becomes stronger. The O-H bonds of the protein side chain undergo similar changes. On the other hand, M formation further weakens the interaction of the same water molecules in BR. The appearance of a sharp band at 3486 cm-1, which was assigned tentatively to the N-H stretching vibration of the peptide bond, is unique to L. The results suggest that the water molecules are involved in the perturbation of Asp-96 in the L intermediate and that they are exerted from the protonated Schiff base which changes position upon the light-induced reaction.     

Components of the carbonyl stretching band in the infrared spectra of hydrated 1,2-diacylglycerolipid bilayers: a reevaluation.

Previous vibrational spectroscopic studies of solid acyl-alkyl and diacyl phosphatidylcholines suggested that the sn1- and sn2-carbonyl stretching modes of 1,2-diacylglycerolipids have different absorption maxima. To address the assignment of sn1- and sn2-carbonyl stretching modes of hydrated 1,2-diacylglycerolipids, aqueous dispersions of 1-palmitoyl-2-hexadecyl phosphatidylcholine (PHPC), 1-hexadecyl-2-palmitoyl phosphatidylcholine (HPPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), as well as hydrated samples of unlabeled, sn1-13C=O-labeled, sn2-13C=O-labeled, and doubly 13C=O-labeled dimyristoylphosphatidylcholine (DMPC) were examined by Fourier transform infrared spectroscopy. The ester carbonyl stretching (nu C=O) bands of HPPC and PHPC each exhibit maxima near 1726 cm-1 and appear to be a summation of three subcomponents with maxima near 1740 cm-1, 1725 and 1705-1711 cm-1. In contrast, the nu C=O band of DPPC exhibits its maximum near 1733 cm-1 and appears to be a summation of two components centered near 1742 and 1727 cm-1. Thus the ester carbonyl group of the acyl-alkyl PCs appears to reside in a more polar environment than the ester carbonyl groups of their diacyl analogue. This observation implies that the polar/apolar interfaces of hydrated bilayers formed by PHPC and by HPPC are significantly different from that of DPPC and raises the question of whether the acyl-alkyl PCs are suitable models of their diacyl analogue. The absorption maximum of the nu C=O band of the doubly 13C=O-labeled DMPC occurs near 1691 cm-1 and those of its subcomponents occur near 1699 and 1685 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox process of iron-sulfur clusters of the soluble-domain of the membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F studied by resonance Raman spectroscopy

Resonance Raman spectra of the soluble-domain of a membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F were recorded in different oxidation states. In the oxidized state, the Raman band due to the totally symmetric stretching mode of the iron-sulfur cluster was observed at 341 cm-1, which was attributed to the 3Fe-4S cluster. In the hydrogen-reduced state, only a weak and broad band was observed in its vicinity. During the process of reoxidation, a Raman band assignable to the 4Fe-4S cluster was observed at 333 cm-1 in the first step. Then, the band at 341 cm-1 became stronger and eventually dominated the spectrum. Corresponding changes were observed in the visible absorption spectra of the same sample. It was concluded from these observations that this hydrogenase has both 3Fe-4S and 4Fe-4S clusters and takes on at least three oxidation states, namely, oxidized, intermediate, and hydrogen-reduced ones.     

A Fourier-transform infrared spectroscopic study of the phosphoserine residues in hen egg phosvitin and ovalbumin

A Fourier-transform infrared spectroscopic study of hen egg phosvitin and ovalbumin has been carried out. Bands arising from monoanionic and dianionic phosphate monoester [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498] can be identified easily in the 1300-930 cm-1 region in spectra of solutions of O-phosphoserine and phosvitin, a highly phosphorylated protein. On the other hand, spectra of ovalbumin show a relatively strong absorption above 1000 cm-1 arising from the protein moiety. Below 1000 cm-1, a single band at 979 cm-1 is observed; this band is not present in spectra of dephosphorylated ovalbumin, and therefore, it has been assigned to the symmetric stretching of the phosphorylated Ser-68 and Ser-344 in the dianionic ionization state. In addition, bands arising from symmetric and antisymmetric stretchings of the monoanionic ionization state, and from the antisymmetric stretching of the dianionic state, can be detected above 1000 cm-1 in difference spectra of ovalbumin minus dephosphorylated ovalbumin. The effect of pH on the infrared spectra of O-phosphoserine, phosvitin, and ovalbumin is consistent with the phosphoserine residues undergoing ionization with pK values about 6. This study demonstrates that Fourier-transform infrared spectroscopy can be a useful technique to assess the ionization state of phosphoserine residues in proteins in solution.     

Fourier transform infrared spectroscopy of 13C = O-labeled phospholipids hydrogen bonding to carbonyl groups

Fourier transform infrared spectroscopy has been used to characterize the carbonyl stretching vibration of DMPC, DMPE, DMPG, and DMPA, all labeled with 13C at the carbonyl group of the sn-2 chain. Due to the vibrational isotope effect, the 13C = O and the 12C = O vibrational bands are separated by ca. 40-43 cm-1. This frequency difference does not change when the labeling is reversed with the 13C = O group at the sn-1 chain. For lipids in organic solvents possible conformational differences between the sn-1 and sn-2 ester groups have no effect on the vibrational frequency of the C = O groups. In aqueous dispersion unlabeled phospholipids always show a superposition of two bands for the C = O vibration located at ca. 1740 and 1727 cm-1. These two bands have previously been assigned to the sn-1 and sn-2 C = O groups. FT-IR spectra of 13C-labeled phospholipids show that the vibrational bands of both, the sn-1 as well as the sn-2 C = O group, are clearly superpositions of at least two underlying components of different frequency and intensity. Band frequencies were determined by Fourier self-deconvolution and second-derivative spectroscopy. The difference between the component bands is ca. 11-17 cm-1. Again, the conformational effect as shown by reversed labeling is negligible with only 1-2 cm-1. The splitting of the C = O vibrational bands in H2O and D2O is caused by hydrogen bonding of water molecules to both C = O groups as shown by a comparison with spectra of model ester compounds in different solvents. To extract quantitative information about changes in hydration, band profiles were stimulated with Gaussian-Lorentzian functions. The chemical nature of the head group and its electronic charge have distinctive effects on the extent of hydration of the carbonyl groups. In the gel and liquid-crystalline phase of DMPC the sn-2 C = O group is more hydrated than the sn-1 C = O. This is accord with the conformation determined by X-ray analysis. In DMPG the sn-1 C = O group seems to be more accessible to water, indicating a different conformation of the glycerol backbone.     

Binding of transducin and transducin-derived peptides to rhodopsin studies by attenuated total reflection-Fourier transform infrared difference spectroscopy.

Fourier transform infrared difference spectroscopy combined with the attenuated total reflection technique allows the monitoring of the association of transducin with bovine photoreceptor membranes in the dark. Illumination causes infrared absorption changes linked to formation of the light-activated rhodopsin-transducin complex. In addition to the spectral changes normally associated with meta II formation, prominent absorption increases occur at 1735 cm-1, 1640 cm-1, 1550 cm-1, and 1517 cm-1. The D2O sensitivity of the broad carbonyl stretching band around 1735 cm-1 indicates that a carboxylic acid group becomes protonated upon formation of the activated complex. Reconstitution of rhodopsin into phosphatidylcholine vesicles has little influence on the spectral properties of the rhodopsin-transducin complex, whereas pH affects the intensity of the carbonyl stretching band. AC-terminal peptide comprising amino acids 340-350 of the transducin alpha-subunit reproduces the frequencies and isotope sensitivities of several of the transducin-induced bands between 1500 and 1800 cm-1, whereas an N-terminal peptide (aa 8-23) does not. Therefore, the transducin-induced absorption changes can be ascribed mainly to an interaction between the transducin-alpha C-terminus and rhodopsin. The 1735 cm-1 vibration is also seen in the complex with C-terminal peptides devoid of free carboxylic acid groups, indicating that the corresponding carbonyl group is located on rhodopsin.     

O-H stretching vibration in Fourier transform difference infrared spectra of bacteriorhodopsin

FTIR difference spectroscopic studies of M intermediate and LA bacteriorhodopsin in the O-H stretching region show bands at 3671 and 3641 cm-1, respectively. The O-H stretching bands in this region may reflect protonation-deprotonation changes or environmental change in the tyrosine residues in bR.     

Two applications of the thermogram of the alcohol/water binary system with compositions of cryobiological interests

Quantitative analyses of the bound water content in the alcohol aqueous solution and its osmotic behavior should be cryobiologically significant. This paper has presented two applications of the thermogram of the alcohol/water system recorded by differential scanning calorimeter (DSC). Both applications are: (1) generating the quantitative relationship between the bound water content and the solution composition; (2) calculating the osmotic virial coefficients for alcohols. Five alcohols including methanol, ethanol, ethylene glycol, propylene glycol and glycerol are investigated. In the present study, partial binary phase diagrams of these five alcohol solutions are determined in the first place. The bound water contents in these solutions are quantitatively evaluated by three criteria afterwards. In the end, the osmotic virial coefficients for these alcohols are calculated according to the osmotic virial equation. It is turned out that the bound water fraction out of the total water content increases with a rising molality. The ability of the solute to restrict water molecules can be weakened when the solution becomes more concentrated. The results also indicate that propylene glycol should be the strongest “water-blocker” while methanol the weakest one. These findings can deepen our understanding of the cryoprotective properties of the alcohols from the perspectives of their roles in binding free water and promoting the osmotic efflux of cell water.