Changes In Infrared Absorption Research Articles

Ion-Protein Interaction in Channel and Pump Proteins Studied by FTIR Spectroscopy

Membrane proteins play crucial roles for receiving external stimuli, transporting and permeating protons and ions to generate biological energy and signal. The molecular mechanisms of membrane proteins have been deepened with the progress in structural biology. Especially, the structural changes connecting the inactive and active states of protein are nowadays investigated by many physicochemical methods. Fourier-transform infrared (FTIR) spectroscopy is one of powerful method for investigating molecular structures in the level of chemical bonds. Perfusion-induced difference infrared spectroscopy has been applied to extract structural changes of protein in response to various stimuli. The selectivity of ion is important for understanding the molecular mechanisms of the ion channels and transporters. I have been studying a sodium ion pump, V-type ATPase [1], and a potassium ion channel, KcsA [2] by using FTIR spectroscopy with attenuated total reflection (ATR) configuration. KcsA is a well-known potassium ion channel whose precise X-ray structures in various conditions have been reported. I have applied ATR-FTIR spectroscopy on KcsA and succeeded to assign the C=O stretching vibrations of the selectivity filter in the amide I region [2]. Recently, I proposed a novel rapid buffer-exchange system for time-resolved ATR-FTIR spectroscopy to monitor ion-binding reaction of protein. The anion-binding reaction of a membrane protein, Natronomonaspharaonis halorhodopsin was confirmed by an increase of the retinal absorption band at 1528 cm−1 in accordance with the buffer-exchange reaction finished within ∼25 ms, which was monitored by the infrared absorption change of a nitrate band at 1350 cm−1 [3].[1] Y. Furutani et al. J. Am. Chem. Soc.133 (9), 2860-3, 2011.[2] Y. Furutani et al. J. Phys. Chem. Lett.3, 3806-10, 2012.[3] Y. Furutani et al. BIOPHYSICS9, 123-129, 2013.

Development of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode.

Attenuated total reflectance (ATR)-FTIR spectroscopy has been widely used to probe protein structural changes under various stimuli, such as light absorption, voltage change, and ligand binding, in aqueous conditions. Time-resolved measurements require a trigger, which can be controlled electronically; therefore, light and voltage changes are suitable. Here we developed a novel, rapid buffer-exchange system for time-resolved ATR-FTIR spectroscopy to monitor the ligand- or ion-binding re-action of a protein. By using the step-scan mode (time resolution; 2.5 ms), we confirmed the completion of the buffer-exchange reaction within ∼25 ms; the process was monitored by the infrared absorption change of a nitrate band at 1,350 cm−1. We also demonstrated the anion-binding reaction of a membrane protein, Natronomonas pharaonis halorhodopsin (pHR), which binds a chloride ion in the initial anion-binding site near the retinal chromophore. The formation of chloride- or nitrate-bound pHR was confirmed by an increase of the retinal absorption band at 1,528 cm−1. It also should be noted that low sample consumption (∼1 µg of protein) makes this new method a powerful technique to understand ligand–protein and ion–protein interactions, particularly for membrane proteins.

Infrared absorption change in single-walled carbon nanotubes observed by combination spectroscopy of synchrotron radiation and laser

The Drude tail due to photo-excited carriers in single-walled carbon nanotubes (SWNTs) has been observed in the mid-infrared region by using combination spectroscopy of synchrotron radiation and Ti:sapphire laser. It is found that the density of photo-excited carriers increases as the sample temperature is raised from 12 to 300 K, and their lifetime is of the order of minutes at 300 K. These facts suggest that the movement of photo-excited carriers is largely affected by some extrinsic defect, thus resulting in the long-lasting Drude reflection in SWNTs.

Secondary Structure and Pd (II) Coordination in S-Layer Proteins from Bacillus sphaericus Studied by Infrared and X-Ray Absorption Spectroscopy

The S-layer of Bacillus sphaericus strain JG-A12, isolated from a uranium-mining site, exhibits a high metal-binding capacity, indicating that it may provide a protective function by preventing the cellular uptake of heavy metals and radionuclides. This property has allowed the use of this and other S-layers as self-assembling organic templates for the synthesis of nanosized heavy metal cluster arrays. However, little is known about the molecular basis of the metal-protein interactions and their impact on secondary structure. We have studied the secondary structure, protein stability, and Pd (II) coordination in S-layers from the B. sphaericus strains JG-A12 and NCTC 9602 to elucidate the molecular basis of their biological function and of the metal nanocluster growth. Fourier transform infrared spectroscopy reveals similar secondary structures, containing ∼35% β-sheets and little helical structure. pH-induced infrared absorption changes of the side-chain carboxylates evidence a remarkably low pK < 3 in both strains and a structural stabilization when Pd (II) is bound. The COO −-stretching absorptions reveal a predominant Pd (II) coordination by chelation/bridging by Asp and Glu residues. This agrees with XANES and EXAFS data revealing oxygens as coordinating atoms to Pd (II). The additional participation of nitrogen is assigned to side chains rather than to the peptide backbone. The topology of nitrogen- and carboxyl-bearing side chains appears to mediate heavy metal binding to the large number of Asp and Glu in both S-layers at particularly low pH as an adaptation to the environment from which the strain JG-A12 has been isolated. These side chains are thus prime targets for the design of engineered S-layer-based nanoclusters.

The phase transition temperatures of a liquid crystal determined from FT-IR spectra explored by principal component analysis

The FT-IR spectra of a thin layer of pure 4-chloro-2′-hydroxy-4′-pentyloxyazobenzene (CHPAB) were studied as a function of temperature. A detailed analysis of the intensity variations was performed by a method based on principal component analysis (PCA). It was shown that the phase transition temperatures obtained by means of PCA and those determined by differential scanning calorimetry (DSC), the most widely used technique in the field, were nearly identical. The PCA results revealed that the transition from solid to a liquid crystalline (LC) phase (smectic A) is more drastic phase transition in terms of infrared absorption changes. The nematic to isotropic phase transition is much less infrared sensitive. Very much smaller absorption changes are associated with the transition between the smectic and nematic mesophases. The pattern of the intensity changes strictly is correlated with the orientation of the CHPAB molecules towards the surface windows due to the surface-induced homeotropic alignment of LC molecules. The important role of hydrogen bonding interaction on the observed transition is disclosed.

Development of Infrared Electroabsorption Spectroscopy and its Application to Molecular Structural Studies

We have constructed an infrared electroabsorption spectroscopic system employing the alternating current (AC) coupled dispersive method developed previously in our laboratory. It is highly sensitive, measuring electric-field-induced infrared absorption changes as small as 6 x 10(-8) in the mid-infrared region of 4000-820 cm(-1). Infrared electroabsorption spectra of liquids and solutions can be measured at room temperature. The system was first applied to liquid acetone to observe the orientational as well as the electronic polarization signals. These signals show distinct behaviors that are exactly predicted by theory. It was then applied to liquid 1,2-dichloroethane, for which electric-field-induced gauche/trans equilibrium changes were detected as infrared absorption changes. The gauche/trans ratio in room-temperature liquid 1,2-dichloroethane has been determined for the first time as 1.4 +/- 0.2. This equilibrium constant has led to the free energy difference deltaG of 0.9 +/- 0.4 kJ mol(-1) (deltaG = G(g) - G(t)) and the entropy difference of deltaS = -3.0 +/- 1.4 J K(-1) mol(-1) with the known enthalpy difference deltaH approximately 0.0 kJ mol(-1). Finally, the system was applied to 1,4-dioxane solutions of N-methylacetamide. The dipole moments of the monomer as well as that of the dimer of N-methylacetamide have been determined separately and a head-to-tail structure of the dimer has been clarified. The instrumentation and the theoretical basis of infrared electroabsorption spectroscopy are described in detail together with the above-mentioned experimental results.

A Fourier transform infrared absorption difference spectrum associated with the reduction of A1 in photosystem I: are both phylloquinones involved in electron transfer?

Photoaccumulated Fourier transform infrared difference spectra associated with P700(+) and P700(+)A(1)(-) formation have been obtained using purified photosystem I particles from Synechocystis sp. PCC 6803. From these spectra, a difference spectrum associated with phylloquinone reduction (A(1)(-) - A(1)) has been calculated. Infrared absorption changes associated with both the loss of the ground state and formation of the anion radical are observed in the difference spectrum. Fourier transform infrared difference spectra obtained in various spectral regions indicate that two, structurally distinct phylloquinones are photoaccumulated. This could indicate that phylloquinones on both the PsaA and PsaB branches are involved in electron transfer, and that electron transfer is bi-directional in photosystem I. It could also indicate an intrinsic structural heterogeneity in the A(1) binding site of the active branch. Several FTIR difference features taken together indicate that a glutamic acid residue (at position 699 or 702 on PsaA and/or 679 or 682 on PsaB) is perturbed upon A(1) anion formation. It is suggested that the protonation state of the perturbed glutamic acid residue is influenced by hydrogen bonding to a nearby tyrosine residue at position 696/676 on PsaA/PsaB.

Binding of Transducin and Transducin-Derived Peptides to Rhodopsin Studied 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 D 2O 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. A C-terminal peptide comprising amino acids 340–350 of the transducin α-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- α 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.

Time-Resolved, Step-Scan FTIR Spectroscopy of Excited States of Transition Metal Complexes

Abstract Time-resolved, step-scan Fourier transform infrared spectroscopy has been developed as a method of studying electronically excited states of transition metal complexes. The technique takes advantage of the unique properties of carbonyl and cyanide stretching vibrations, including high infrared oscillator strength and well established sensitivity of vibrational frequency, intensity, and bandwidth to electronic and molecular structure. Electronic excitation generally produces significant transient infrared absorption changes which are characteristic of the changes in electronic structure, such as oxidation of the metal. TRIR spectroscopy thus provides new insight on the nature of the excited state transition (e.g. charge transfer vs. ligand centered), the extent of charge transfer, communication between metal centers, and energy and electron transfer processes. The step-scan FTIR approach has significant advantages over conventional time-resolved techniques, including spectral multiplexing, increased IR throughput, and fast data acquisition, making it possible to rapidly obtain complete spectra with good sensitivity and time-resolution.

Picosecond infrared studies of the dynamics of the photosynthetic reaction center.

The changes in the vibrational transitions of the protein and redox cofactors of the photosynthetic reaction center were examined by picosecond infrared spectroscopy. The spectra in the vibrational mid-infrared region (1800-1550 cm-1) of hydrated and partially dehydrated reaction centers were investigated from 50 ps to 4 ns after photoinitiation of the electron transfer. Features in the infrared difference spectra were identified with both protein and redox cofactor vibrational modes and correlated with electron transfer events whose kinetics were measured in the infrared and visible regions. The observed protein response is confined to a few amide I transitions (1644 cm-1, 1661 cm-1, 1665 cm-1) and carboxylic residues (1727 cm-1). About 85% of the observed signal corresponded to alterations in the cofactor-associated ester and keto carbonyls. The amide I and carboxylic transitions appeared prior to 50 ps, suggesting that the primary electron transfer event is coupled with a specific piece of the protein backbone and to glutamic or aspartic residues nearby the special pair. Infrared absorption changes accompanying bacteriochlorophyll-dimer cation formation dominated the signal at all times investigated. Infrared spectral changes observed in hydrated and partially dehydrated reaction centers were distinctly different; a band at 1665 cm-1 with a spectral width of 6 cm-1 in the hydrated protein, corresponding to a protein amide I bleach, was not present in the dehydrated film. These differences are discussed in terms of the markedly different electron transfer kinetics observed in the presence of water.

Molecular changes following oxidoreduction of cytochrome b559 characterized by Fourier transform infrared difference spectroscopy and electron paramagnetic resonance: photooxidation in photosystem II and electrochemistry of isolated cytochrome b559 and iron protoporphyrin IX-bisimidazole model compounds.

The vibrational infrared absorption changes associated with the oxidation of cytochrome b559 (Cyt b559) have been characterized. In photosystem II (PS II) enriched membranes, low-potential (LP) and high-potential (HP) Cyt b559 were investigated by light-induced FTIR difference spectroscopy. The redox transition of isolated Cyt b559 is characterized by protein electrochemistry. On the basis of a model of the assembly of Cyt b559 with the two axial Fe ligands being histidine residues of two distinct polypeptides, each forming a transmembrane alpha-helix [Cramer, W.A., Theg, S.M., & Widger, W.R. (1986) Photosynth. Res. 10, 393-403], the bisimidazole and bismethylimidazole complexes of Fe protoporphyrin IX were electrochemically oxidized and reduced to detect the IR oxidation markers of the heme and its two axial ligands. Major bands at 1674/1553, 1535, and 1240 cm-1 are tentatively assigned to nu 37 (CaCm), nu 38-(CbCb) and delta (CmH) modes, respectively; other bands at 1626, 1613, 1455, 1415, and 1337 cm-1 are assigned to porphyrin skeletal and vinyl modes. Modes at 1103 and 1075/1066 cm-1 are assigned to the 4-methylimidazole and imidazole ligands, respectively. For the isolated Cyt b559, it is shown that both the heme (at 1556-1535, 1337, and 1239 cm-1), the histidine ligands at 1104 cm-1 and the protein (between 1600 and 1700 cm-1 and at 1545 cm-1) are affected by the charge stabilization. The excellent agreement between model compounds and isolated Cyt b559 reinforces the validity of the model of a heme iron coordinated to two histidine residues for Cyt b559. A differential signal at 1656/1641 cm-1 is assigned to peptide C = O mode(s). We speculate that this signal reflects the change in strength of a hydrogen bond formed between the histidine ligand(s) and the polypeptide backbone upon oxidoreduction of the cytochrome. In PS II membranes, the signals characteristic of Cyt b559 photooxidation are found at 1660/1652 and 1625 cm-1, for both the high- and low-potential forms. The differences observed in the amplitude of the 1660/1652-cm-1 band, at 1700 and 1530-1510 cm-1 in the light-induced FTIR difference spectra of Cyt b559 HP and LP, show that the mechanisms of heme oxidation in vivo imply different molecular processes for the two forms Cyt b559 HP and LP.

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates.

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.

The effects of heat treatment on dislocation-free oxygen-containing silicon crystals

A dislocation-free silicon crystal grown by the Czochralski technique has been heat treated over a wide temperature-time range to explore the basic silicon-oxygen relationship. The processes occurring were followed by carrier-concentration changes and infrared measurements over the range 1.2–25 μ. The infrared absorption changes occurring below 900 °C were found to follow an Arrhenius plot with an activation energy of 0.5±0.03 eV. This value is compared with the results found for other processes occurring on the surface or in bulk silicon and is seen to be low. The changes occurring above 900 °C are in agreement with the generally accepted theory that all the oxygen initially present in the crystal returns to its bound interstitial position. The carrier-concentration changes measured were at variance with currently accepted theories in several respects, particularly at 650 °C, an important temperature since all silicon producers use it to stabilize resistivity for measurement control prior to the thermal cycling experienced during device manufacture. Several experiments are described where slices were thermally cycled to study some of the ’’equilibrium’’ carrier-concentration situations believed to hold. Finally, a general discussion is given of the overall situation concerning the behavior of the various silicon-oxygen complexes believed to exist at the various temperatures. Some speculation is presented as to the interactions of the various Si-O complexes, but it is found that all the results cannot be fully explained on the basis of purely silicon-oxygen interactions and that other effects, particularly those due to carbon, must be taken into consideration.

Light-induced spectral shifts in bacteriochlorophyll and carotenoid absorption in purple bacteria

Light-induced absorbancy changes in the near-infrared wavelength region in intact cells and cell free preparations of photosynthetic bacteria have been object of several investigations ( Duysens, 1952; Duysens et al., 1956; Arnold and Clayton, 1960; Clayton, 1962; Vredenberg and Duysens, 1963; Sybesma and Vredenberg, 1963, 1964; Kuntz et al., 1964). An absorption decrease around 870–890 mμ in purple bacteria has been attributed to the oxidation of a reaction center P 890 upon illumination. Recently Clayton (1963) reported in Rhodopseudomonas spheroides a different type of infrared absorption changes. These changes, which are not observed in Rhodospirillum rubrum (e.g. Duysens et al., 1956), could not be attributed to the oxidation of P 890, because of their kinetics and sign at 890 mμ. This paper gives the results of a closer study of the characteristics of these changes in various species of sulphur and non sulphur purple bacteria. In contradistinction to Clayton we conclude that they are not caused by a primary light reaction.

AN INFRARED AND ISOPIESTIC INVESTIGATION OF THE INTERACTION BETWEEN TRI-n-BUTYL PHOSPHATE AND MONO-(2-ETHYLHEXYL)-PHOSPHORIC ACID1

The interaction between tri-n-butyl phosphate and mono(2-ethylhexyl)- phosphonic acid was investigated. The method of continuous variations utilizing changes in infra red absorption and changes in the freezing point depression of the mixtures was used. Isopiestic measurements also were made. Results gave evidence that an association product had resulted at a ratio of 2 moles of TBP per hexamer unit of H/sub 2/MEHP. (auth)