LIGHT-INDUCED PERTURBATION OF AROMATIC RESIDUES IN BOVINE RHODOPSIN AND BACTERIORHODOPSIN*

Light-induced changes in the UV absorption spectrum of bovine rod outer segment membranes were measured by conventional difference spectroscopy and by flash photolysis methods. Different thermal intermediates of rhodopsin (lumirhodopsin, metarhodopsin I, metarhodopsin II, and meta-rhodopsin III) have absorption spectra in the ultraviolet which differ from the rhodopsin spectrum and from each other. The spectra associated with metarhodopsin I, metarhodopsin II, and metarhodopsin III are characteristic of perturbation of a small number of tyr. and/or trp residues, most likely one trp residue. These aromatic residues are in the neighborhood of the retinal Schiff base and undergo coordinated changes of interaction with retinal during the bleaching sequence. At the metarhodopsin II stage, the magnitude of the UV spectral changes is consistent with the exposure of a previously shielded trp residue to an aqueous environment. The present results are consistent with previous spectral studies which limit the extent of light-induced conformational changes to regions of the protein in the neighborhood of the retinal Schiff base. An analogous study was made on light-adapted purple membranes of Halobacterium halobium. The UV absorption spectrum associated with the deprotonated Schiff base intermediate of the trans-bacteriorhodopsin cycle is indicative, in part, of aromatic residue perturbation. However, significant changes in the secondary and tertiary structures of the bacterio-rhodopsin protein characteristic of a delocalized conformational change are unlikely at this intermediate stage.     

SPIN LABELED CYSTEINES AS SENSORS FOR PROTEIN-LIPID INTERACTION AND CONFORMATION IN RHODOPSIN

In stoichiometric amounts, the spin label N-tempoyl-(p-chloromercuribenzamide) reacts rapidly with one cysteine residue in membrane-bound bovine rhodopsin. This residue is distinct from the two reactive cysteines previously used as attachment sites for spectroscopic labels, and is on the external surface of the protein near the cytoplasmic membrane/aqueous interface. The spin-labeled side chain has revealed a light-induced conformational change in membrane-bound rhodopsin that is apparently not associated with protein aggregation. The changes are reversible upon the addition of 11-cis retinal, and the magnitude of the change is dependent on the identity of the phospholipid in the surrounding bilayer. Alteration of lipid composition has a much larger effect on bleached rhodopsin than rhodopsin itself, indicating that the former is more readily deformable in response to changes in bilayer properties. This is consistent with the loss of 11-cis retinal binding energy in opsin compared to rhodopsin. These results provide direct structural evidence that the conformation of a membrane protein can be modulated by the lipid properties.     

LIGHT-INDUCED CHANGE IN RHODOPSIN EMISSION: PHOSPHORESCENCE and FLUORESCENCE

Abstract— Phosphorescence measurements of rhodopsin in bovine rod disk membranes were made to study changes in protein conformation on bleaching by probing the environment of tryptophan and tyrosine residues of the protein. Bleaching decreased the tyrosine phosphorescence by about 25% and significantly affected the amplitude of triplet decay when rhodopsin was excited at 280 nm, where both tyrosine and tryptophan absorb. Computer analysis using one or two exponential model functions showed the presence of two components in the decay curve at 410 nm—one with a lifetime of 2.2 s, the other with a lifetime of 4.8 s>—which are typical of tyrosine and tryptophan respectively. When the rod outer segment sample was bleached, there was a significant decrease in the amplitude of the tyrosine component. However, the lifetime values of the two components did not change. Analyses of the fluorescence spectra of dark and bleached membranes at different excitation wavelengths and the phosphorescence change on bleaching suggest energy transfer between tyrosine and tryptophan singlet states, which may result from a conformational change of the opsin moiety on bleaching.     

Real time observation of proteolysis with Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy: watching a protease eat a protein

Fourier transform infrared (FT-IR)- and UV-circular dichroism (UV-CD) spectroscopy have been used to study real-time proteolytic digestion of β-lactoglobulin (β-LG) and β-casein (β-CN) by trypsin at various substrate/enzyme ratios in D(2)O-buffer at 37°C. Both techniques confirm that protein substrate looses its secondary structure upon conversion to the peptide fragments. This perturbation alters the backbone of the protein chain resulting in conformational changes and degrading of the intact protein. Precisely, the most significant spectral changes which arise from digestion take place in the amide I and amide II regions. The FT-IR spectra for the degraded β-LG show a decrease around 1634 cm(-1), suggesting a decrease of β-sheet structure in the course of hydrolysis. Similarly, the intensity around the 1654 cm(-1) band decreases for β-CN digested by trypsin, indicating a reduction in the α-helical part. On the other hand, the intensity around ～1594 cm(-1) and ～1406 cm(-1) increases upon enzymatic breakdown of both substrates, suggesting an increase in the antisymmetric and symmetric stretching modes of free carboxylates, respectively, as released digestion products. Observation of further H/D exchange in the course of digestion manifests the structural opening of the buried groups and accessibility to the core of the substrate. On the basis of the UV-CD spectra recorded for β-LG and β-CN digested by trypsin, the unordered structure increases concomitant with a decrease in the remaining structure, thus, revealing breakdown of the intact protein into smaller fragments. This model study in a closed reaction system may serve as a basis for the much more complex digestion processes in an open reaction system such as the stomach.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

The effects of stretching on wool fibres as monitored by FT-Raman spectroscopy

FT-Raman spectroscopy coupled with amide I band deconvolution was used to monitor the conformational changes of the peptide backbone of sulphite pre-treated wool fibres during stretching. The spectral changes observed are consistent with the transition of -helical to β-pleated sheet structure. These changes, which are most rapid during the early stages of stretching, can be related to protein secondary structure at both the crystalline and molecular levels. Analysis of the amide III region of the spectra reveals that a very small amount of additional disorder is imparted to the peptide backbone as a result of stretching. The consistency in the widths at half-height of the amide I band components implies that stretching does not significantly change the distribution of peptide chain conformations. From the Raman analysis of cells isolated from the stretched fibres, it is evident that conformational changes occur in both the cuticle and cortex. The most evident change, however, is in the cortical cells.     

Effects of light and cGMP on membrane fluidity of frog rod outer segments.

Abstract—We have used a spin-labeled fatty acid to detect changes induced by light and by cGMP in isolated rod outer segment membranes. We chose a spin probe (5-doxylstearic acid) which has the nitroxide group placed on the hydrocarbon chain, so the probe should reside somewhat inside the hydrophobic region of the membrane. We found that light exposures which bleached the rhodopsin also produced a small change in the EPR spectra. The spectral changes are consistent with a small increase in membrane fluidity. Light exposures which bleach rhodopsin are known to activate a phosphodiesterase that markedly decreases the cGMP level in rod outer segment. Therefore, we attempted to vary cGMP levels directly by adding Bu₂-cGMP, or indirectly, by adding IBMX, CDTA or ATP to try to inhibit the phosphodiesterase. In each case where the cGMP level is expected to increase, we observed spectral changes in the dark which suggested a small decrease in membrane fluidity. Thus, all of our results with this probe are consistent with the idea that changing the level of cGMP produces changes in membrane fluidity. The light-induced spectral changes we observed required the presence of ATP, and were inhibited by 2mM Ca²⁺, or by the chelator of divalent cations, CDTA.     

Carbon-13 NMR spectroscopic studies on chemically modified and unmodified synthetic and natural humic acids

Carbon-13 cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy ((13)C-CP/MAS-NMR) was applied to study the chemical modification process of humic acids (HA) with diazomethane and the subsequent alkaline hydrolysis of the methylated HA. This modification process results in HA with selectively blocked phenolic OH groups, which can be used for metal ion binding studies with humic substances. Different chemically modified and unmodified natural and synthetic HA with carbon-13 of natural abundance were investigated. In addition, carbon-13 labeled modified synthetic HA, that were synthesized with [(13)C]diazomethane as methylation reagent, were studied to confirm the assumed modification process and to determine the type of functional groups that have the highest affinity for methylation with diazomethane. The results of the NMR studies with carbon-13 labeled modified HA show that predominantly carboxyl and phenolic OH groups are methylated with diazomethane resulting in methyl ester and methyl ether groups, respectively. Due to the alkaline treatment of the methylated HA, the methyl esters of carboxyl groups are hydrolyzed, whereas methyl ethers of phenolic OH groups remain unchanged, which results in modified HA with blocked phenolic OH groups. From the spectra of the modified and unmodified HA with carbon-13 of natural abundance it can be concluded that the applied preparative modification procedure causes only the desired structural changes in HA.     

Fluorescence polarization studies of B-phycoerythrin oriented in polymer film

Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured. The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models. The interactions between various chromophores of the pigment-protein complexes investigated were discussed. The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation. This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both). Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission. Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions. It shows that several fractions of differently oriented "forms" of chromophores exhibiting different spectral properties occur in B-PE complexes. Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration. Based on the results obtained a new reorientational mechanism of the State 1 --> State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET. Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 --> State 2 transition). The proposed mechanism needs further verification by the investigation of whole bacteria cells.     

碳酸钙的原位合成及表面改性

利用碳化法, 选用几种常见的改性剂(硬脂酸钠、十八碳醇磷酸酯和油酸)对碳酸钙进行了原位合成及表面改性. 通过活化度、白度、接触角的测定, 对比了其改性效果, 同时通过傅里叶变换红外光谱(FT-IR)、X射线衍射(XRD)、透射电镜(TEM)等测试手段对产品进行表征. FT-IR结果表明, 改性剂与碳酸钙表面是以化学键合和物理吸附方式相结合. 碳酸钙改性后, 其红外υ3特征吸收峰出现约44 cm-1的蓝移现象. 对反应机理进行了初步探讨. 实验结果表明, 当十八碳醇磷酸酯用量达到2%(以碳酸钙的质量分数计)时, 产品活化度达到99.9%, 白度值达到97.3%, 接触角达到了122.25°, 从而为新型无机填料的制备提供了理论依据和合成手段.     

Adsorptive fouling of modified and unmodified commercial polymeric ultrafiltration membranes

The fouling tendency, due to adsorption on the pore walls, of two pairs of modified and unmodified ultrafiltration membranes, with similar observed retentions determined by dextran and gel permeation chromatography, was studied. The membranes investigated were made of modified and unmodified polyaramide (PA) and modified and unmodified polyvinylidene fluoride (PVDF). The PVDF membrane was surface-modified and the PA membrane was made from a modified polymer solution. Membrane modification was used to reduce fouling by adsorption. Octanoic acid was used as the fouling substance, representing a large number of small, hydrophobic compounds. It is demonstrated in this investigation that membrane modification is not always successful. It was determined that at lower concentrations of octanoic acid, the modified PA membrane exhibits a smaller fouling tendency than the unmodified PA membrane, while the result is reversed for concentrations above 60% of the saturation concentration. The fouling tendency of the unmodified PVDF membrane is much lower than that of the modified PVDF membrane at all concentrations. The cross-sections of the membranes were visually examined with scanning electron microscopy, but no difference could be observed between the modified and unmodified membranes. The membranes were also examined with Fourier transform infrared spectroscopy. The spectra of the two PA membranes were different, while no difference was observed for the unmodified and surface-modified PVDF membranes. Remains of octanoic acid were found in the membranes, although they had been thoroughly rinsed with deionized water and the initial pure water flux was recovered.     

Vibrational spectra and molecular conformation of diethyl fumarate and especially the maleate

Infrared and Raman spectra of diethyl maleate and diethyl fumarate were recorded in the liquid and solid states. From the spectral changes on phase transition the known conformational equilibrium s-trans/s-cis ? s-trans/s-trans in the case of dialkyl fumarates is confirmed, and the view that dialkyl maleates exist only in a single conformation is corrected. The observation of several bands in the vCC region of IR and Raman spectra of diethyl maleate, and significant intensity shifts in this region with phase transition prove the existence of at least two conformers in liquid dialkyl maleates.     

环氧树脂水基化化学改性的研究

用对氨基苯甲酸改性环氧树脂 ,使其成为具有亲水性的树脂。测定了改性树脂的溶解性 ,发现改性后树脂在有机溶剂中的溶解性能变差 ,但在碱性溶剂中溶解性增强。对改性树脂进行了红外光谱表征 ,并根据环氧基特征峰的吸收对环氧基转化率进行了定量分析。测定了改性产物的DSC曲线 ,发现随着反应物中对氨基苯甲酸比例的提高 ,改性产物的玻璃化转变温度升高。涂膜的性能测试表明 ,对氨基苯甲酸改性环氧树脂水基涂料的机械力学性能和耐化学试剂性能比溶剂型纯环氧树脂要优越。     

Photoluminescence Properties of Surface-Modified Nanocrystalline ZnS : Mn

DBS surface-modified nanocrystalline ZnS : Mn was prepared by a precipitation method. Photoluminescence spectra and decay curves were measured and compared to those of unmodified samples. For both kinds of samples, luminescence decay curves of 600 nm emission consist of two components with nanosecond (ns) and millisecond (ms) lifetimes. The nanosecond component is attributed to zinc vacancies luminescence, with peak located at 440 nm. While the millisecond component is attributed to Mn(2+) luminescence and its decay time changes with Mn(2+) concentration and surface modification. The surface-modified sample has a longer lifetime compared to unmodified samples. The photoaging of surface-modified samples is slow. Particle size effects on photoaging were also examined. For nanocrystalline ZnS : Mn photoaging is more noticeable than that of bulk material. After UV irradiation, photoluminescence intensity of the modified sample is several times larger than that of the unmodified sample, because the surface-active reagent decreases surface defects and depresses radiationless transitions. XPS yields direct evidence of the surface-modification effect on the surface structure. Copyright 2000 Academic Press.     

Influence of bi modification of pt anode catalyst in direct formic acid fuel cells

The influence of Bi modification of Pt anode catalyst on the performance of direct formic acid fuel cells was investigated. Compared with the unmodified Pt anode, the Bi modified Pt (PtBi(m)) electrode prepared by under-potential deposition (UPD) caused faster electrocatalytic oxidation of formic acid at the same value of the overpotential, and thus, PtBi(m) resulted in an increase in the power performance of direct formic acid fuel cells. Electrochemical impedance spectra helped to explain the difference of performance between the unmodified Pt and Bi modified Pt electrodes. Solution conductivity and dehydration phenomena occurring in highly concentrated formic acid solutions can also explain the higher power performance of PtBi(m).     

Insights from first principles molecular dynamics studies toward infrared multiple-photon and single-photon action spectroscopy: case study of the proton-bound dimethyl ether dimer

We have used finite temperature ab initio molecular dynamics simulations in conjunction with computation of critical quantum nuclear effects to probe the differences between single-photon argon tagged action spectral results and infrared multiple-photon dissociation experiments for a proton bound molecular ion system. We find that the principal difference between the results in these experimental techniques is essentially that of cluster temperature. The multiple-photon dissociation experiments conducted using room temperature ions reflect a larger degree of conformational freedom compared to the colder single-photon argon tagged action spectral results. Our ab initio molecular dynamics simulation techniques accurately capture the effects of conformational sampling, adequately reproduce both spectra, and can be utilized to assign the dynamically averaged finite temperature spectra.     

DFT and IR spectroscopic analysis of p-tert-butylthiacalix[4]arene

Vibrational analysis of p-tert-butylthiacalix[4]arene based on an experimental mid and far IR spectra is described here. Density functional calculations of a most stable cone conformer related to crystalline solid FT-IR spectra in the temperature range up to 180 degrees C as well spectra of dilute solution have been used to obtain a better understanding of conformational state and a hydrogen bonding of p-tert-butylthiacalix[4]arene. Complete assignments were made for experimental FT-IR spectra of the cone conformer. Heating (up to 180 degrees C) of crystalline p-tert-butylthiacalix[4]arene did not change both conformational and cooperative H-bonding state of its molecules. A temperature spectral effect seems to be due to some relaxation of crystal packing mostly viewed on the OH bands.     

Assigning vibrational spectra of ferryl-oxo intermediates of cytochrome C oxidase by periodic orbits and molecular dynamics

Complexity is inherent in biological molecules not only because of the large number of atoms but also because of their nonlinear interactions responsible for chaotic behaviours, localized motions, and bifurcation phenomena. Thus, versatile spectroscopic techniques have been invented to achieve temporal and spatial resolution to minimize the uncertainties in assigning the spectra of complex molecules. Can we associate spectral lines to specific chemical bonds or species in a large molecule? Can energy stay localized in a bond for a substantial period of time to leave its spectroscopic signature? These longstanding problems are investigated by studying the resonance Raman spectra of ferryl-oxo intermediates of cytochrome c oxidase. The difference spectra of isotopically substituted ferryl oxygen ((16)O minus (18)O) in the cytochrome c oxidase recorded in several laboratories show one or two prominent positive peaks which have not been completely elucidated yet. By applying the hierarchical methods of nonlinear mechanics, and particularly the study of periodic orbits in the active site of the enzyme, in conjunction with molecular dynamics calculations of larger systems which include the embraced active site by the protein and selected protonated/deprotonated conformations of amino acids, we translate the spectral lines to molecular motions. It is demonstrated that for the active site stable periodic orbits exist for a substantial energy range. Families of periodic orbits which are associated with the vibrations of Fe(IV)=O bond mark the regions of phase space where nearby trajectories remain localized, as well as assign the spectral bands of the active site in the protein matrix. We demonstrate that proton movement adjacent to active site, which occurs during the P --> F transition, can lead to significant perturbations of the Fe(IV)=O isotopic difference vibrational spectra in cytochrome c oxidase, without a change in oxidation state of the metal sites. This finding links spectroscopic characteristics to protonation events occurring during enzymatic turnover.     

LIGHT-INDUCED CONFORMATIONAL CHANGES IN CATTLE RHODOPSIN AS PROBED BY MEASUREMENTS OF THE INTEFFACE POTENTIAL

Abstract— A novel technique of capacitative coupling of oriented rhodopsin at a polar/apolar interface allows the time resolved investigation of conformational changes following a flash. Electric signals arise as a consequence of changes of the interface potential. A signal occurring within milliseconds behaves like the R₂-phase of the "early receptor potential" (= ERP). This response is interpreted as a conformational change of rhodopsin. No correlation of this signal is found to the spectroscopically defined metarhodopsin I-metarhodopsin II transition.
The temperature dependence of the conformational change coincides with the temperature dependence of the latency of the 'a-wave' of the electroretinogram, reported by Arden and Ikeda (1968). It is suggested that the command step of visual excitation is the conformational change and not one of the spectroscopically defined photolysis steps of rhodopsin.
Analysis of slower electrical signals following the fast response is in accordance with the model of a light-induced pore formation.     

L-arginine trifluoroacetate salt bridges in its solid state compound: the low-temperature three dimensional structural determination of L-arginine bis(trifluoroacetate) crystal and its vibrational spectral analysis

Structural varieties of L-arginine trifluoroacetate (abbreviated as LATF) and L-arginine bis(trifluoroacetate), LABTF, in the solid state compounds were observed and analyzed by the nuclear magnetic resonance (NMR) spectroscopy. The guanidinium-carboxylate interaction plays an important role involving in the crystal structure construction. Conformational changes of L-Arg(+) and L-Arg(2+) cations result from the intrinsic structural difference by hydrogen bonding and electrostatic interactions. The low-temperature structure of its crystalline salt, L-arginine bis(trifluoroacetate), was determined to describe the hydrogen bonding interactions. In comparison with the crystal structure at room temperature, the low-temperature L-Arg(2+) cations present tiny conformational difference and the rotational disorder of CF(3) group disappears. FT-IR and Raman spectra were investigated and hydrogen bonding interactions were analyzed on the basis of its vibrational spectra. Results indicate that this type interaction is greatly contributive to the structural features and vibrational spectral properties.