Two progressive substrates of the M-intermediate can be identified in glucose-embedded, wild-type bacteriorhodopsin.

Glucose-embedded bacteriorhodopsin shows M-intermediates with different Amide I infrared bands when samples are illuminated at 240 or 260 K, in contrast with fully hydrated samples where a single M-intermediate is formed at all temperatures. In hydrated, but not in glucose-embedded specimens, the N intermediate is formed together with M at 260 K. Both Fourier transform infrared and electron diffraction data from glucose-embedded bacteriorhodopsin suggest that at 260 K a mixture is formed of the M-state that is trapped at 240 K, and a different M-intermediate (MN) that is also formed by mutant forms of bacteriorhodopsin that lack a carboxyl group at the 96 position, necessary for the M to N transition. The fact that an MN species is trapped in glucose-embedded, wild-type bacteriorhodopsin suggests that the glucose samples lack functionally important water molecules that are needed for the proton transfer aspartate 96 to the Schiff base (and, thus, to form the N-intermediate); thus, aspartate 96 is rendered ineffective as a proton donor.     

Conformational changes of bacteriorhodopsin detected by Fourier transform infrared difference spectroscopy

We report the first Fourier transform infrared difference spectra of purple membrane. Evidence is presented that alterations in the vibrations of both the retinylidene chromophore and the protein groups of bacteriorhodopsin associated with photocycling can be detected. This method provides a new tool for probing the conformational changes occurring in bacteriorhodopsin during the proton pump cycle.     

Microwave-Induced Structural Changes in Bacteriorhodopsin: Studies by Optical and Fourier Transform Infrared Difference Spectroscopy

Biophysics - Abstract—Using optical and Fourier transform infrared (FTIR) difference spectroscopy, microwave radiation was found to affect the bacteriorhodopsin (BR) structure in films at a...     

Bacteriorhodopsin's M412 and BR605 protein conformations are similar Significance for proton transport

Cation removal or acidification induces a transition of bacteriorhodopsin (BR₅₆₈) to a blue species (BR₆₀₅). Fourier transform infrared spectroscopy now reveals that the protein conformational changes accompanying this transition resemble those which occur during the BR₅₆₈ to M₄₁₂ transition and associated light-driven proton transport. This and other results suggest that BR₆₀₅ and M₄₁₂ both have similar voltage-dependent protein conformations in which a secondary structure is stabilized by a change in the local electric potential sensed by internal charged groups involved in proton transport.     

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals.

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold. In this paper, we use Fourier transform infrared spectroscopy to characterize the secondary structure of highly purified native and proteolyzed AQP1 reconstituted in membrane crystalline arrays and compare it to bacteriorhodopsin. For this analysis the fractional secondary structure contents have been determined by using several different algorithms. In addition, a neural network-based evaluation of the Fourier transform infrared spectra in terms of numbers of secondary structure segments and their interconnections [sij] has been performed. The following conclusions were reached: 1) AQP1 is a highly helical protein (42-48% alpha-helix) with little or no beta-sheet content. 2) The alpha-helices have a transmembrane orientation, but are more tilted (21 degrees or 27 degrees, depending on the considered refractive index) than the bacteriorhodopsin helices. 3) The helices in AQP1 undergo limited hydrogen/deuterium exchange and thus are not readily accessible to solvent. Our data support the AQP1 structural model derived from sequence prediction and epitope insertion experiments: AQP1 is a protein with at least six closely associated alpha-helices that span the lipid membrane.     

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~10²-10³ repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10^-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.     

Polarized infrared spectroscopy of oriented purple membrane.

Polarized Fourier transform infrared spectroscopy has been used to study the structure of purple membrane from Halobacterium halobium. Membranes were oriented by drying a suspension of membrane fragments onto Irtran-4 slides. Dichroism measurements of the amide I, II and A peaks were used to find the average spatial orientation of the bacteriorhodopsin alpha-helices. By deriving a function that relates the observed dichroism to the orientational order parameters for the peptide groups, helical axis distribution, and mosaic spread of the membranes, the average orientation of the alpha-helices was found to lie in a range of less than 26 degrees away from the membrane normal, agreeing with electron microscopic measurements. The frequency of the amide I and A peaks is at least 10 cm-1 higher than values found for most alpha-helical polypeptides and proteins. This may indicate that bacteriorhodopsin contains distorted alpha-helical conformations.     

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.     

Fourier transform infrared analysis of bacteriorhodopsin secondary structure.

Fourier transform infrared spectroscopy at a resolution of 1 cm-1 has been used to study the conformation of dark-adapted bacteriorhodopsin in the native purple membrane, in H2O and D2O suspensions. A detailed analysis of the amide I bands was made using derivative and deconvolution techniques. Curve-fitting results of four independent experiments indicate, after estimation of the methodological errors, that native bacteriorhodopsin contains 52-73% alpha-helices, 13-19% reverse turns, 11-16% beta-sheets, and 3-7% unordered segments. Our analysis has enabled the identification of several components corresponding to alpha-helices, beta-sheets, and reverse turns. Besides the alpha I- and alpha II-helices (peaking at 1658 and 1665 cm-1), we propose that two more infrared bands arise from alpha-helical structures: one at 1650 cm-1 from alpha I and another one at 1642 cm-1 in H2O suspension, which could originate from type III beta-turns (i.e., one turn of 3(10)-helix). The relatively high content of reverse turns suggests the presence of one reverse turn per loop, plus another one in the C-terminal segment. On the other hand, several reasons argue that the calculated mean beta-sheet content of around 14% should be decreased somewhat. These beta-sheets could be located in the noncytoplasmatic links of the bacteriorhodopsin molecule.     

脱脂菌紫质与天然紫膜水合变化的比较

本实验通过不同水合度下天然紫膜、脱脂菌紫质吸附等温线分析、红外光谱对比,讨论了天然紫膜小磷脂、蛋白质、水三者作用关系,认为磷脂对天然紫膜中蛋白质表而一些极性基团的分布及水合有重要作用,这些位点的水合对蛋白质进一步水合变化起重要作用.     

Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure.

The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferlé, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.     

Evidence for light-induced lysine conformational changes during the primary event of the bacteriorhodopsin photocycle

Fourier transform infrared difference spectroscopy is used to examine the role of lysine in the primary event of the bacteriorhodopsin photocycle. Isotopically labeled lysine is used to tentatively assign the lysine modes in the BR and K species. The results suggest that the lysine side-chain undergoes conformational changes in concert with the known light-induced chromophore structural alterations.     

Temperature Dependence of Water Interactions with the Amide Carbonyls of α-Helices

Hydration is a key determinant of the folding, dynamics, and function of proteins. In this study, temperature-dependent Fourier transform infrared (FTIR) spectroscopy combined with singular value decomposition (SVD) and global fitting were used to investigate both the interaction of water with α-helical proteins and the cooperative thermal unfolding of these proteins. This methodology has been applied to an isolated α-helix (Fs peptide) and to globular α-helical proteins including the helical subdomain and full-length villin headpiece (HP36 and HP67). The results suggest a unique IR signature for the interaction of water with the helical amide carbonyl groups of the peptide backbone. The IR spectra indicate a weakening of the net hydrogen bond strength of water to the backbone carbonyls with increasing temperature. This weakening of the backbone solvation occurs as a discrete transition near the maximum of the temperature-dependent hydrophobic effect, not a continuous change with increasing temperature. Possible molecular origins of this effect are discussed with respect to previous molecular dynamics simulations of the temperature-dependent solvation of the helix backbone.     

Vibrational spectroscopy of seaweed galactans

Information from classical infrared spectroscopy studies has been of significance for characterizing seaweed galactans. The development of Fourier transform infrared spectroscopy and of Fourier transform laser Raman spectroscopy has produced great advances in the application of vibrational spectroscopy to the structural study of polysaccharides. Computational facilities in the spectrometers allow the arithmetic manipulations of the spectra. The second-derivative mode in the FT IR spectrocopy provided more information by increasing the number and resolution of the bands in the spectra as compared to the parent ones. A review of literature data on vibrational spectroscopy of sulfated polysaccharides and new results are presented. Agar-type polymers showed two diagnostic bands in the second-derivative mode in the region 800–700 cm^–1. Carrageenans exhibited a number of bands in the region 1600–1000 cm^–1. Fourier transform laser Raman spectroscopy in the solid state gave well-defined characteristic spectra of agar and carrageenans. Both techniques can be applied to small samples in the solid state and allow differentiation in a few minutes between agar and carrageenan-type seaweed galactans. The second-derivative mode of the FT IR spectra can be applied to distinguish agar-producing from carrageenan-producing seaweeds. The spectra on KBr pellets of dried, ground agarophyte and carrageenophyte seaweed samples showed the same bands as the corresponding polysaccharides.     

Effect of heavy metals on tissue protein of an edible fish Cirrhinus mrigala as dependent on pH and water hardness

The goal of the study was to investigate the influence of nickel and chromium at different pH and water hardness on the protein content of muscle tissues of Cirrhinus mrigala fingerlings by Fourier transform infrared spectroscopy. FTIR spectra revealed significant differences in absorbance intensities between control and metal-exposed muscle tissues, reflecting a change in protein content and state caused by heavy metal toxicity; metal accumulation in tissue was markedly increased at alkaline pH and to a lesser extent in hard water.     

Substitution of amino acids in helix F of bacteriorhodopsin: effects on the photochemical cycle

The effects of amino acid substitutions in helix F of bacteriorhodopsin on the photocycle of this light-driven proton pump were studied. The photocycles of Ser-183----Ala and Glu-194----Gln mutants were qualitatively similar to that of wild-type bacteriorhodopsin produced in Escherichia coli and bacteriorhodopsin from Halobacterium halobium. The substitution of a Phe for either Trp-182 or Trp-189 significantly reduced the fraction of photocycling bacteriorhodopsin. The amino acid substitutions Tyr-185----Phe and Ser-193----Ala substantially increased the lifetime of the photocycle without substantially increasing the lifetime of the M photocycle intermediate. Similar results were also obtained with the Pro-186----Gly substitution. In contrast, replacing Pro-186 with the larger residue Leu inhibited the formation of the M photocycle intermediate. These results are consistent with a structural model of the retinal-binding pocket suggested by low-temperature UV/visible and Fourier transform infrared difference spectroscopies that has Trp-182, Tyr-185, Pro-186, and Trp-189 forming part of the binding pocket.     

Molecular structure and vibrational spectra of 4-nitrobenzylchloride by ab initio Hartree–Fock and density functional methods

The Fourier transform Raman and Fourier transform infrared spectra 4-nitrobenzylchloride of (NBC) were recorded in the solid phase. The Fourier transform gas phase infrared spectrum of NBC was also recorded. The equilibrium geometry, harmonic vibrational frequencies, infrared intensities and Raman scattering activities were calculated by HF/DFT (B3LYP and BLYP) and SVWN methods with the 6-31G(d,p) basis set. The scaled theoretical wave numbers by B3LYP showed very good agreement with the experimental ones. A detailed interpretation of the infrared and Raman spectra of NBC is reported on the basis of the calculated potential energy distribution. The theoretical spectrograms for the IR spectrum of the title molecule have been constructed.     

Evidence for a controlling role of water in producing the native bacteriorhodopsin structure.

The experiments reported in this paper, based on reconstitution of bacteriorhodopsin (bR) from apomembrane at varying environmental conditions, demonstrate that the presence of water is a controlling factor in generating a native wild-type bR conformation. If water is lacking during this reconstitution process, then a non-native bR structure is formed that exhibits altered M formation and decay kinetics, as well as different behavior following extensive dehydration. It is shown that mutants affecting the ability of bR to form appropriate structures of water in specific protein cavities also affect the ability to generate a native bR conformation. The results suggest that aspartic acid 96 plays a major role in anchoring the appropriate water structure conformation associated with bR. It is also demonstrated that the glutamic acid 204 residue is pivotal in controlling the protein/water affinity. This water affinity can be further controlled by modifying the charge environment of the protein with altered pH. These data, based on kinetic absorption spectroscopy and Fourier transform infrared spectroscopy, highlight the central role of water in this protein.     

Analysis of conformational changes in bacteriorhodopsin upon retinal removal.

The conformation of bacterioopsin in the apomembrane has been studied by Fourier transform infrared spectroscopy. Resolution enhancement techniques and curve-fitting procedures have been used to determine the secondary structural components from the amide I region. Bacterioopsin contains about 54% helicoidal structure (alpha I and alpha II helices + 3(10) turns), 21% sheets, 16% reverse turns, and 9% unordered structure. Thus, after retinal removal, all of the secondary structural types of bacteriorhodopsin remain present, and only slight quantitative differences appear. On the other hand, H/D exchange studies show that there is a higher degree of exchange for reverse turns and protonated carboxylic lateral chains in bacterioopsin as compared to bacteriorhodopsin. This gives further support to the idea of a more open tertiary structure of bacterioopsin, and to the consideration of the retinal molecule as an important element in complementing the interhelical interactions in bacteriorhodopsin folding.     

Synthesis and characterization of a stable dispersion of multifunctional Gd2O3:Er,Yb in polyvinyl alcohol

A stable dispersion of multifunctional Gd₂O₃:Er,Yb phosphor in polyvinyl alcohol (PVA) was synthesized by varying the concentration of Yb³⁺ ions. It had a strong ultraviolet–visible‐near infrared (UV–vis) upconversion emission and applications in temperature and magnetic field sensors (e.g., nano‐heaters), as well as potential use in bioleveling and bioimaging. Stability of the dispersion was found to strongly depend on the mixing process of the powder in the polymer solution. Spherical shaped nanoparticles in cubic phase of ~ 43 nm diameter were synthesized and characterized by X‐ray diffraction; results were confirmed by scanning electron microscopy (SEM). Fourier transform infrared sspectroscopy (FT‐IR) and thermal analysis supported the presence of PVA. NIR pumping produced strong UC emission bands in the red and green regions extending up to very high UV (240 nm). This method provides an alternative for synthesizing a highly UC‐efficient non‐agglomerated pure transparent dispersion from various efficient phosphors for biological applications. Copyright © 2012 John Wiley & Sons, Ltd.