Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy

Crystalline structures of cellulose (named as Cell 1), NaOH-treated cellulose (Cell 2), and subsequent CO2-treated cellulose (Cell 2-C) were analyzed by wide-angle X-ray diffraction and FTIR spectroscopy. Transformation from cellulose I to cellulose II was observed by X-ray diffraction for Cell 2 treated with 15-20 wt% NaOH. Subsequent treatment with CO2 also transformed the Cell 2-C treated with 5-10 wt% NaOH. Many of the FTIR bands including 2901, 1431, 1282, 1236, 1202, 1165, 1032, and 897 cm(-1) were shifted to higher wave number (by 2-13 cm(-1)). However, the bands at 3352, 1373, and 983 cm(-1) were shifted to lower wave number (by 3-95 cm(-1)). In contrast to the bands at 1337, 1114, and 1058 cm(-1), the absorbances measured at 1263, 993, 897, and 668 cm(-1) were increased. The FTIR spectra of hydrogen-bonded OH stretching vibrations at around 3352 cm(-1) were resolved into three bands for cellulose I and four bands for cellulose II, assuming that all the vibration modes follow Gaussian distribution. The bands of 1 (3518 cm(-1)), 2 (3349 cm(-1)), and 3 (3195 cm(-1)) were related to the sum of valence vibration of an H-bonded OH group and an intramolecular hydrogen bond of 2-OH ...O-6, intramolecular hydrogen bond of 3-OH...O-5 and the intermolecular hydrogen bond of 6-O...HO-3', respectively. Compared with the bands of cellulose I, a new band of 4 (3115 cm(-1)) related to intermolecular hydrogen bond of 2-OH...O-2' and/or intermolecular hydrogen bond of 6-OH...O-2' in cellulose II appeared. The crystallinity index (CI) was obtained by X-ray diffraction [CI(XD)] and FTIR spectroscopy [CI(IR)]. Including absorbance ratios such as A1431,1419/A897,894 and A1263/A1202,1200, the CI(IR) was evaluated by the absorbance ratios using all the characteristic absorbances of cellulose. The CI(XD) was calculated by the method of Jayme and Knolle. In addition, X-ray diffraction curves, with and without amorphous halo correction, were resolved into portions of cellulose I and cellulose II lattice. From the ratio of the peak area, that is, peak area of cellulose I (or cellulose II)/total peak area, CI(XD) were divided into CI(XD-CI) for cellulose I and CI(XD-CII) for cellulose II. The correlation between CI(XD-CI) (or CI(XD-CII)) and CI(IR) was evaluated, and the bands at 2901 (2802), 1373 (1376), 897 (894), 1263, 668 cm(-1) were good for the internal standard (or denominator) of CI(IR), which increased the correlation coefficient. Both fraction of the absorbances showing peak shift were assigned as the alternate components of CI(IR). The crystallite size was decreased to constant value for Cell 2 treated at >or= 15 wt% NaOH. The crystallite size of Cell 2-C (cellulose II) was smaller than that of Cell 2 (cellulose I) treated at 5-10 wt% NaOH. But the crystallite size of Cell 2-C (cellulose II) was larger than that of Cell 2 (cellulose II) treated at 15-20 wt% NaOH.     

Two-dimensional/ATR infrared correlation spectroscopic study on water diffusion in a poly(epsilon-caprolactone) matrix

In the present contribution, two-dimensional ATR-FTIR spectroscopy was used to study the diffusion of water in poly(epsilon-caprolactone) (PCL). In the spectral region of the nu(OH) stretching vibration of water, four absorption bands (3635, 3560, 3411, and 3220 cm(-1)) can be identified. The higher wavenumber band pair at 3635 and 3560 cm(-1) is assigned to the antisymmetric and symmetric OH stretching vibrations, respectively, of water which is partially hydrogen-bonded to the carbonyl groups of PCL, whereas the lower frequency band pair at 3411 (antisymmetric) and 3220 cm(-1) (symmetric) is attributed to the OH stretching vibrations of bulk water which is fully hydrogen-bonded to other water molecules. From the asynchronous map of a 2D correlation analysis of spectra recorded during the diffusion of water into PCL, it was concluded that during the diffusion process the water molecules first penetrate into the free volume (microvoids) of the PCL matrix or are molecularly dispersed in the polymer matrix and then form hydrogen bonds with the C=O groups of the polymer.     

Ca2+ release from caged-Ca2+ alters the FTIR spectrum of sarcoplasmic reticulum

Light-induced Ca2+ release from the Ca2+ complex of Nitr-5 altered the FTIR spectra of sarcoplasmic reticulum vesicles and purified Ca(2+)-ATPase preparations. The principal changes seen in difference spectra obtained after and before illumination in the presence of Nitr-5.Ca2+ consisted of an increase in absorbance at 1663 and 1676 cm-1 and a decrease in absorbance at 1653 cm-1. The light-induced changes in FTIR spectra were prevented by vanadate or EGTA, indicating that they were associated with the formation of Ca2E1 enzyme intermediate. Other light-induced changes in the FTIR spectra at 1600-1250 cm-1 were not clearly related to the sarcoplasmic reticulum, and were attributed to photolysis of Nitr-5. The difference absorbance bands are narrow, suggesting that they originate from changes in side chain vibrations, although some changes in secondary structures may also contribute.     

FTIR detection of water reactions in the oxygen-evolving centre of photosystem II

Flash-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the water-oxidizing reactions in the oxygen-evolving centre of photosystem II. Reactions of water molecules were directly monitored by detecting the OH stretching bands of weakly H-bonded OH of water in the 3700-3500 cm(-1) region in FTIR difference spectra during S-state cycling. In the S1-->S2 transition, a band shift from 3588 to 3617 cm(-1) was observed, indicative of a weakened H-bond. Decoupling experiments using D2O:H2O (1:1) showed that this OH arose from a water molecule with an asymmetric H-bonding structure and this asymmetry became more significant upon S2 formation. In the S2-->S3, S3-->S0 and S0-->S1 transitions, negative bands were observed at 3634, 3621 and 3612 cm(-1), respectively, representing formation of a strong H-bond or a proton release reaction. In addition, using complex spectral features in the carboxylate stretching region (1600-1300 cm-(1)) as 'fingerprints' of individual S-state transitions, pH dependency of the transition efficiencies and the effect of dehydration were examined to obtain the information of proton release and water insertion steps in the S-state cycle. Low-pH inhibition of the S2-->S3, S3-->S0 and S0-->S1 transitions was consistent with a view that protons are released in the three transitions other than S1-->S2, while relatively high susceptibility to dehydration in the S2-->S3 and S3-->S0 transitions suggested the insertion of substrate water into the system during these transitions. Thus, a possible mechanism of water oxidation to explain the FTIR data is proposed.     

Water-mediated hydrogen-bonded network on the cytoplasmic side of the Schiff base of the L photointermediate of bacteriorhodopsin

The L intermediate in the proton-motive photocycle of bacteriorhodopsin is the starting state for the first proton transfer, from the Schiff base to Asp85, in the formation of the M intermediate. Previous FTIR studies of L have identified unique vibration bands caused by the perturbation of several polar amino acid side chains and several internal water molecules located on the cytoplasmic side of the retinylidene chromophore. In the present FTIR study we describe spectral features of the L intermediate in D(2)O in the frequency region which includes the N-D stretching vibrations of the backbone amides. We show that a broad band in the 2220-2080 cm(-1) region appears in L. By use of appropriate (15)N labeling and mutants, the lower frequency side of this band in L is assigned to the amides of Lys216 and Gly220. These amides are coupled to each other, and interact with Thr46 and Val49 in helix B and Asp96 in helix C via weakly H-bonding water molecules that exhibit O-D stretching vibrations at 2621 and 2605 cm(-1). These water molecules are part of a hydrogen-bonded network characteristic of L which includes other water molecules located closer to the chromophore that exhibit an O-D stretching vibration at 2589 cm(-1). This structure, extending from the Schiff base to the internal proton donor Asp96, stabilizes L and affects the L-to-M transition.     

Molecular changes during tensile deformation of single wood fibers followed by Raman microscopy

Raman spectra were acquired in situ during tensile straining of mechanically isolated fibers of spruce latewood. Stress-strain curves were evaluated along with band positions and intensities to monitor molecular changes due to deformation. Strong correlations (r = 0.99) were found between the shift of the band at 1097 cm(-1) corresponding to the stretching of the cellulose ring structure and the applied stress and strain. High overall shifts (-6.5 cm(-1)) and shift rates (-6.1 cm(-1)/GPa) were observed. After the fiber failed, the band was found on its original position again, proving the elastic nature of the deformation. Additionally, a decrease in the band height ratio of the 1127 and 1097 cm(-1) bands was observed to go hand in hand with the straining of the fiber. This is assumed to reflect a widening of the torsion angle of the glycosidic C-O-C bonding. Thus, the 1097 cm(-1) band shift and the band height ratio enable one to follow the stretching of the cellulose at a molecular level, while the lignin bands are shown to be unaffected. Observed changes in the OH region are shown and interpreted as a weakening of the hydrogen-bonding network during straining. Future experiments on different native wood fibers with variable chemical composition and cellulose orientation and on chemically and enzymatically modified fibers will help to deepen the micromechanical understanding of plant cell walls and the associated macromolecules.     

Characterization of uniaxially aligned chitin film by 2D FT-IR spectroscopy

Two-dimensional FT-IR spectroscopy (2D FT-IR) was applied to the investigations of crystalline chitin structure. From this study, new information about spectral bands which are not observed in conventional 1D FT-IR was obtained. In 2D spectra, three specific bands were differentiated at 3482, 3421, and 3380 cm(-1) in the OH region. They could be assigned as C(6)OH groups which are hydrogen-bonded to the next C(6)OH; C(3)OH groups hydrogen-bonded to O(5); and C(6)OH groups bifurcated hydrogen-bonded to C(6)OH as well as C=O, respectively. Two pairs of bands appeared in the amide region, indicating the two types of hydrogen-bonded states of C=O groups. This is in good agreement with the results in the OH region; half of the C(6)OH groups are hydrogen-bonded to C=O as well as the next C(6)OH. The results accurately confirmed the Blackwell model which was established by an X-ray diffraction study. The temperature-dependency of hydrogen-bonds was also revealed by 2D FT-IR. Interchain hydrogen-bonds [C(6)OH...O(6)] first respond to a temperature followed by intrachain [C(6)OH...O=C] and [C(3)OH...O(5)] with increasing temperature. Interchain hydrogen-bonds [NH...O=C] are relatively stable in the temperature range of 40-180 degrees C.     

A conformational intermediate between the resting and desensitized states of the nicotinic acetylcholine receptor

The structural changes induced in the nicotinic acetylcholine receptor by two noncompetitive channel blockers, proadifen and phencyclidine, have been studied by infrared difference spectroscopy and using the conformationally sensitive photoreactive noncompetitive antagonist 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine. Simultaneous binding of proadifen to both the ion channel pore and neurotransmitter sites leads to the loss of positive markers near 1663, 1655, 1547, 1430, and 1059 cm(-)(1) in carbamylcholine difference spectra, suggesting the stabilization of a desensitized conformation. In contrast, only the positive markers near 1663 and 1059 cm(-)(1) are maximally affected by the binding of either blocker to the ion channel pore suggesting that the conformationally sensitive residues vibrating at these two frequencies are stabilized in a desensitized-like conformation, whereas those vibrating near 1655 and 1430 cm(-)(1) remain in a resting-like state. The vibrations at 1547 cm(-)(1) are coupled to those at both 1663 and 1655 cm(-)(1) and thus exhibit an intermediate pattern of band intensity change. The formation of a structural intermediate between the resting and desensitized states in the presence of phencyclidine is further supported by the pattern of 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine photoincorporation. In the presence of phencyclidine, the subunit labeling pattern is distinct from that observed in either the resting or desensitized conformations; specifically, there is a concentration-dependent increase in the extent of photoincorporation into the delta-subunit. Our data show that domains of the nicotinic acetylcholine receptor interconvert between the resting and desensitized states independently of each other and suggest a revised model of channel blocker action that involves both low and high affinity agonist binding conformational intermediates.     

Characterization of the CuA center in the cytochrome c oxidase from Thermus thermophilus for the spectral range 1800-500 cm-1 with a combined electrochemical and Fourier transform infrared spectroscopic setup

In this study we present the electrochemically induced Fourier transform infrared (FTIR) difference spectra of the Cu(A) center derived from the ba(3)-type cytochrome c oxidase of Thermus thermophilus in the spectral range from 1800 to 500 cm(-1). The mid infrared is dominated by the nu(C[double bond]O) vibrations of the amide I modes at 1688, 1660, and 1635 cm(-1), reflecting the redox-induced perturbation of the predominantly beta-sheet type structure. The corresponding amide II signal is found at 1528 cm(-1). In the lower frequency range below 800 cm(-1), modes from amino acids liganding the Cu(A) center are expected. On the basis of the absorbance spectrum of the isolated amino acids, methionine is identified as an important residue, displaying C-S vibrations at these frequencies. This spectral range was previously disregarded by protein IR spectroscopists, mainly due to the strong absorbance of the solvent, H(2)O. With an optimized setup, however, IR is found suitable for structure/function studies on proteins.     

Infrared spectra and hydrogen bonding of pentitols and pyranosides at 20 to 300 K

Infrared spectra in the range 400-4000 cm(-1) of three pentitols--ribitol, xylitol, D-arabinitol, and of three pyranosides--methyl alpha-D-manno-, methyl alpha-D-gluco- and methyl beta-D-galactopyranoside, as polycrystalline solids of both the pure OH and > 90% isotopically substituted OD compounds, were recorded at 20-300 K. In the low temperature spectra of the OH substances, at least three isolated narrow bands in the stretching mode and about ten narrow bands in the out-of-plane-bending mode range (< 1000 cm(-1)) are affected by cooling. Almost all have counterparts in the respective OD spectra with frequency ratios of 1.30-1.40. On this basis, they are assigned to OH groups bonded in H-bonds of different strengths (from 10 to 50 kJ mol(-1)). The average number of the OH...O hydrogen bonds is found to be two to three times larger than indicated by the stretching mode only or by structural data. The newly measured peak frequencies of the very narrow decoupled stretching mode bands show a correlation between the red shift (delta nu) and the H-bond length. As previously found for tetritols, the presence of weak H-bonds (bond energy < 14 kJ mol(-1)) is related to the different water sorption capabilities of the pentitols.     

Dissecting the chemistry of nicotinic receptor-ligand interactions with infrared difference spectroscopy.

The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm(-1) indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm(-1) are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm(-1) indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm(-1) to 1690 and 1620 cm(-1), respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions.     

Structural changes that occur upon photolysis of the Fe(II)(a3)-CO complex in the cytochrome ba(3)-oxidase of Thermus thermophilus: a combined X-ray crystallographic and infrared spectral study demonstrates CO binding to Cu(B)

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a(3) moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba(3)-oxidase from Thermus thermophilus, determined at ~2.8-3.2? resolution, reveal a Fe-C distance of ~2.0?, a Cu-O distance of 2.4? and a Fe-C-O angle of ~126°. Upon photodissociation at 100K, X-ray structures indicate loss of Fe(a3)-CO and appearance of Cu(B)-CO having a Cu-C distance of ~1.9? and an O-Fe distance of ~2.3?. Absolute FTIR spectra recorded from single crystals of reduced ba(3)-CO that had not been exposed to X-ray radiation, showed several peaks around 1975cm(-1); after photolysis at 100K, the absolute FTIR spectra also showed a significant peak at 2050cm(-1). Analysis of the 'light' minus 'dark' difference spectra showed four very sharp CO stretching bands at 1970cm(-1), 1977cm(-1), 1981cm(-1), and 1985cm(-1), previously assigned to the Fe(a3)-CO complex, and a significantly broader CO stretching band centered at ~2050cm(-1), previously assigned to the CO stretching frequency of Cu(B) bound CO. As expected for light propagating along the tetragonal axis of the P4(3)2(1)2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba(3) crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO(2) at 2337cm(-1) and one from traces of CO at 2133cm(-1); while bands associated with CO bound to either Fe(a3) or to Cu(B) in "light" minus "dark" FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2? and FTIR spectra support the long-held position that photolysis of Fe(a3)-CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu(B) atom; Fe(a3) and Cu(B) ligation, at the resolutions reported here, are otherwise unaltered.     

Alcohols dehydrate lipid membranes: an infrared study on hydrogen bonding.

The effects of alcohols (methanol, ethanol, and n-butanol) on the hydrogen bonding of dipalmitoylphosphatidylcholine (DPPC) were studied by Fourier-transform infrared spectroscopy (FTIR) in water-in-oil (carbon tetrachloride) reversed micelles. The bound O-H stretching mode of water, bonded to DPPC, appeared as a broad band at around 3400 cm-1. The O-H bending mode of this complex appeared as a weak broad band at 1644 cm-1. No free O-H signal was observed. When alcohols were added, a part of DPPC-bound water was replaced by the alcohols. The released 'free' water appeared at 3680 cm-1. This free O-H stretching band represents water-alcohol complex. A new broad band of O-H stretching appeared at 3235 cm-1, which represents the alcohol molecules bound to the phosphate moiety of DPPC. When the alcohol concentration was increased, the intensities of the free O-H stretching and bending bands increased. The P = O- antisymmetric stretching band at 1238 cm-1 became broader and shifted to lower frequencies. This means that alcohols interacted with the phosphate moiety and replaced the bound water. In the deconvoluted spectra of the C = O stretching mode, the ratio between the free sn-2 and the hydrogen-bonded sn-2 bands increased; a part of the bound water at the sn-2 carbon in the glycerol skeleton is also released and the free sn-2 signal increased. From the change in the intensity of the P = O- stretching band, the partition coefficients of alcohols between the phosphate region of DPPC and water were estimated: methanol 7.8, ethanol 16.7 at 22.0 degrees C in mole fraction bases. In molality, these values translates into methanol 0.21 and ethanol 0.45. These results indicate that short-chain alcohols interact with lipid membranes at the phosphate moiety at the hydrophilic head, weaken the membrane-water interaction, and destabilize membranes.     

FTIR studies of internal water molecules in the Schiff base region of bacteriorhodopsin

In a light-driven proton pump protein, bacteriorhodopsin (BR), three water molecules participate in a pentagonal cluster that stabilizes an electric quadrupole buried inside the protein. Previously, low-temperature Fourier-transform infrared (FTIR) difference spectra between BR and the K photointermediate in D(2)O revealed six O-D stretches of water in BR at 2690, 2636, 2599, 2323, 2292, and 2171 cm(-)(1), while five water bands were observed at 2684, 2675, 2662, 2359, and 2265 cm(-)(1) for the K intermediate. The frequencies are widely distributed over the possible range of stretching vibrations of water, and water molecules at <2400 cm(-)(1) were suggested to hydrate negative charges because of their extremely strong hydrogen bonds. In this paper, we aimed to reveal the origin of these water bands in the K minus BR spectra by use of various mutant proteins. The water bands were not affected by the mutations at the cytoplasmic side, such as T46V, D96N, and D115N, implying that the water molecules in the cytoplasmic domain do not change their hydrogen bonds in the BR to K transition. In contrast, significant modifications of the water bands were observed for the mutations in the Schiff base region and at the extracellular side, such as R82Q, D85N, T89A, Y185F, D212N, R82Q/D212N, and E204Q. From these results, we concluded that the six O-D stretches of BR originate from three water molecules, water401, -402, and -406, involved in the pentagonal cluster. Two stretching modes of each water molecule are highly separate (300-470 cm(-)(1) for O-D stretches and 500-770 cm(-)(1) for O-H stretches), which is consistent with the previous QM/MM calculation. The small amplitudes of vibrational coupling are presumably due to strong association of the waters to negative charges of Asp85 and Asp212. Among various mutant proteins, only D85N and D212N lack strongly hydrogen-bonded water molecules (<2400 cm(-)(1)) and proton pumpimg activity. We thus infer that the presence of a strong hydrogen bond of water is a prerequisite for proton pumping in BR. Internal water molecules in such a specific environment are discussed in terms of functional importance for rhodopsins.     

FTIR detection of water reactions during the flash-induced S-state cycle of the photosynthetic water-oxidizing complex

Photosynthetic water oxidation is performed via the light-driven S-state cycle in the water-oxidizing complex (WOC) of photosystem II (PS II). To understand its molecular mechanism, monitoring the reaction of substrate water in each S-state transition is essential. We have for the first time detected the reactions of water molecules in WOC throughout the S-state cycle by observing the OH vibrations of water using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. Moderately hydrated (or deuterated) PS II core films from Synechococcus elongatus were used to obtain the FTIR difference spectra upon the first, second, third, and fourth flash illumination, representing the structural changes in the S(1) --> S(2), S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1) transitions, respectively. In the weakly H-bonded OH region, bands appeared at 3617/3588 cm(-1) as a differential signal in the first-flash spectrum and at 3634, 3621, and 3612 cm(-1) with negative intensities in the second-, third-, and fourth-flash spectra, respectively. These bands shifted down by approximately 940 cm(-1) upon deuteration and by approximately 10 cm(-1) upon H(18)O substitution, indicating that they arise from the OH stretches of water including the substrate and its intermediates. Strongly D-bonded OD bands of water were also identified as broad features in the range of 2600-2200 cm(-1) by taking the double difference between the spectra of D(2)(16)O- and D(2)(18)O-deuterated films. In addition, broad continuum features that probably arise from the large proton polarizability of H-bonds were observed around 3000, 2700, 2550, and 2600 cm(-1) in the first-, second-, third-, and fourth-flash spectra, respectively, of the hydrated PS II film, revealing changes in the H-bond network of the protein. The negative OH intensities upon the second to fourth flashes might be related to proton release from substrate water. The results presented here showed that FTIR detection of water OH(D) bands can be a powerful method for investigating the mechanism of photosynthetic water oxidation.     

Two-dimensional correlation spectroscopy and principal component analysis studies of temperature-dependent IR spectra of cotton-cellulose

The FTIR spectra were measured for raw Uplands Sicala-V2 cotton fibers over a temperature range of 40-325 degrees C to explore the temperature-dependent changes in the hydrogen bonds of cellulose. These cotton-cellulose spectra exhibited complicated patterns in the 3800-2800 cm(-1) region and thus were analyzed by both the exploratory principal component analysis (PCA) and two-dimensional (2-D) correlation spectroscopy methods. The exploratory PCA showed that the spectra separate into two groups on the basis of thermal degradation of the cotton-cellulose and the consequent breakage of intersheet H-bonds present in its structure. Frequency variables, which strongly contributed to each principal component highlighted in its loadings plot, were linked to the frequencies assigned to vibrations of the OH groups involved in different kinds of H-bonds, as well as to vibrations of the CH groups. Deeper insights into reorganization of the temperature-dependent hydrogen bonding were obtained by 2-D correlation spectroscopy. Synchronous and asynchronous spectra were analyzed in the temperature ranges of 40 to 150 and 250 to 320 degrees C, the ranges indicated by PCA. Detailed band assignments of the OH stretching region and changes in the patterns of the hydrogen bonding network of the cotton-cellulose were proposed with the aid of the 2-D correlation spectroscopy analysis. Below 150 degrees C, distinctly different bands assigned to the less stable Ialpha and the more stable Ibeta interchain H-bonds O-6-H-6...O-3' were observed at about 3230 and 3270 cm(-1), respectively. Evaporation of water entrapped in the cellulose network was examined by means of the band at about 3610 cm(-1). The cooperativity of hydrogen bonds, which play a key role in the cellulose conformation, was monitored by frequencies assigned to intrachain H-bonds. It was possible to separate the frequencies assigned to the O-2-H-2...O-6 and O-3-H-3...O-5 intrachain H-bonds into two separate ranges, the spread of which was controlled by the cooperativity effect. The temperature dependence of the asynchronous spectra indicated that the less stable O-3-H-3...O-5 bonds gave rise to an absorption extending from 3300 to 3384 cm(-1), while the more stable O-2-H-2...O-6 bonds were characterized by the absorption between 3400 and 3470 cm(-1). The final breaking of the inter- and intrachain H-bonds, which occurs at the higher temperatures, was monitored by the asynchronous peaks at 3533 and 3590 cm(-1), respectively. On the basis of both the exploratory PCA and 2-D correlation spectroscopy investigations, it was possible to extract well-defined wavenumber ranges assigned to different kinds of intra- and interchain hydrogen bonds, as well as to the free OH groups of the cotton-cellulose.     

Protein-bound water molecules in primate red- and green-sensitive visual pigments

Protein-bound water molecules play crucial roles in the structure and function of proteins. The functional role of water molecules has been discussed for rhodopsin, the light sensor for twilight vision, on the basis of X-ray crystallography, Fourier transform infrared (FTIR) spectroscopy, and a radiolytic labeling method, but nothing is known about the protein-bound waters in our color visual pigments. Here we apply low-temperature FTIR spectroscopy to monkey red (MR)- and green (MG)-sensitive color pigments at 77 K and successfully identify water vibrations using D(2)O and D(2)(18)O in the whole midinfrared region. The observed water vibrations are 6-8 for MR and MG, indicating that several water molecules are present near the retinal chromophore and change their hydrogen bonds upon retinal photoisomerization. In this sense, color visual pigments possess protein-bound water molecules essentially similar to those of rhodopsin. The absence of strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) is common between rhodopsin and color pigments, which greatly contrasts with the case of proton-pumping microbial rhodopsins. On the other hand, two important differences are observed in water signal between rhodopsin and color pigments. First, the water vibrations are identical between the 11-cis and 9-cis forms of rhodopsin, but different vibrational bands are observed at >2550 cm(-1) for both MR and MG. Second, strongly hydrogen-bonded water molecules (2303 cm(-1) for MR and 2308 cm(-1) for MG) are observed for the all-trans form after retinal photoisomerization, which is not the case for rhodopsin. These specific features of MR and MG can be explained by the presence of water molecules in the Cl(-)-biding site, which are located near positions C11 and C9 of the retinal chromophore. The averaged frequencies of the observed water O-D stretching vibrations for MR and MG are lower as the λ(max) is red-shifted, suggesting that water molecules are involved in the color tuning of our vision.     

Strongly hydrogen-bonded water molecule present near the retinal chromophore of Leptosphaeria rhodopsin, the bacteriorhodopsin-like proton pump from a eukaryote

Leptosphaeria rhodopsin (LR) is an archaeal-type rhodopsin found in fungi, and is the first light-driven proton-pumping retinal protein from eukaryotes. LR pumps protons in a manner similar to that of bacteriorhodopsin (BR), a light-driven proton pump of haloarchaea. The amino acid sequence of LR is more homologous to that of Neurospora rhodopsin (NR) than BR, whereas NR has no proton-pumping activity. These facts raise the question of how the proton-pumping function is achieved. In this paper, we studied structural changes of LR following the retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy, and compared the obtained spectra with those for BR and NR. While the light-induced photoisomerization from the all-trans to 13-cis form was commonly observed among LR, BR, and NR, we found that the structural changes of LR are closer to those of BR than to those of NR in terms of detailed vibrational bands of retinal and protein. The most prominent difference was seen for the water O-D stretching vibrations (measured in D2O). LR exhibits an O-D stretch of water at 2257 cm(-1), indicating the presence of a strongly hydrogen-bonded water molecule. Such strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) were observed for BR, but not for NR. Comprehensive studies of BR mutants and archaeal rhodopsins have revealed that strongly hydrogen-bonded water molecules are found only in the proteins exhibiting proton-pumping activity, suggesting that strongly hydrogen-bonded water molecules and transient weakening of their binding are essential for the proton-pumping function of rhodopsins. This observation for LR provided additional experimental evidence of the correlation between strongly hydrogen-bonded water molecules and proton-pumping activity of archaeal rhodopsins.     

Different structural changes occur in blue- and green-proteorhodopsins during the primary photoreaction

We examine the structural changes during the primary photoreaction in blue-absorbing proteorhodopsin (BPR), a light-driven retinylidene proton pump, using low-temperature FTIR difference spectroscopy. Comparison of the light-induced BPR difference spectrum recorded at 80 K to that of green-absorbing proteorhodopsin (GPR) reveals that there are several differences in the BPR and GPR primary photoreactions despite the similar structure of the retinal chromophore and all-trans --> 13-cis isomerization. Strong bands near 1700 cm(-1) assigned previously to a change in hydrogen bonding of Asn230 in GPR are still present in BPR. However, additional bands in the same region are assigned on the basis of site-directed mutagenesis to changes occurring in Gln105. In the amide II region, bands are assigned on the basis of total (15)N labeling to structural changes of the protein backbone, although no such bands were previously observed for GPR. A band at 3642 cm(-1) in BPR, assigned to the OH stretching mode of a water molecule on the basis of H2(18)O substitution, appears at a different frequency than a band at 3626 cm(-1) previously assigned to a water molecule in GPR. However, the substitution of Gln105 for Leu105 in BPR leads to the appearance of both bands at 3642 and 3626 cm(-1), indicating the waters assigned in BPR and GPR exist in separate distinct locations and can coexist in the GPR-like Q105L mutant of BPR. These results indicate that there exist significant differences in the conformational changes occurring in these two types proteorhodopsin during the initial photoreaction despite their similar chromophore structures, which might reflect a different arrangement of water in the active site as well as substitution of a hydrophilic for hydrophobic residue at residue 105.     

Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of Pseudomonas aeruginosa on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm(-1). All strains produced biofilms that absorbed IR radiation near 1,650 cm(-1) (amide I), 1,550 cm(-1) (amide II), 1,240 cm(-1) (P==O stretching, C---O---C stretching, and/or amide III vibrations), 1,100 to 1,000 cm(-1) (C---OH and P---O stretching) 1,450 cm(-1), and 1,400 cm(-1). The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm(-1) (C---OH stretching of alginate) and 1,250 cm(-1) (C---O stretching of the O-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 micrometer, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 micrometer in depth. In contrast, FRD1 produced microcolonies that were approximately 40 micrometer in depth. An algJ mutant strain that produced alginate lacking O-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the algJ mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for P. aeruginosa biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in P. aeruginosa biofilm architecture.