Nanosecond retinal structure changes in K-590 during the room-temperature bacteriorhodopsin photocycle: picosecond time-resolved coherent anti-stokes Raman spectroscopy.

Time-resolved vibrational spectra are used to elucidate the structural changes in the retinal chromophore within the K-590 intermediate that precedes the formation of the L-550 intermediate in the room-temperature (RT) bacteriorhodopsin (BR) photocycle. Measured by picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS), these vibrational data are recorded within the 750 cm-1 to 1720 cm-1 spectral region and with time delays of 50-260 ns after the RT/BR photocycle is optically initiated by pulsed (< 3 ps, 1.75 nJ) excitation. Although K-590 remains structurally unchanged throughout the 50-ps to 1-ns time interval, distinct structural changes do appear over the 1-ns to 260-ns period. Specifically, comparisons of the 50-ps PTR/CARS spectra with those recorded with time delays of 1 ns to 260 ns reveal 1) three types of changes in the hydrogen-out-of-plane (HOOP) region: the appearance of a strong, new feature at 984 cm-1; intensity decreases for the bands at 957 cm-1, 952 cm-1, and 939 cm-1; and small changes intensity and/or frequency of bands at 855 cm-1 and 805 cm-1; and 2) two types of changes in the C-C stretching region: the intensity increase in the band at 1196 cm-1 and small intensity changes and/or frequency shifts for bands at 1300 cm-1 and 1362 cm-1. No changes are observed in the C = C stretching region, and no bands assignable to the Schiff base stretching mode (C = NH+) mode are found in any of the PTR/CARS spectra assignable to K-590. These PTR/CARS data are used, together with vibrational mode assignments derived from previous work, to characterize the retinal structural changes in K-590 as it evolves from its 3.5-ps formation (ps/K-590) through the nanosecond time regime (ns/K-590) that precedes the formation of L-550. The PTR/CARS data suggest that changes in the torsional modes near the C14-C15 = N bonds are directly associated with the appearance of ns/K-590, and perhaps with the KL intermediate proposed in earlier studies. These vibrational data can be primarily interpreted in terms of the degree of twisting of the C14-C15 retinal bond. Such twisting may be accompanied by changes in the adjacent protein. Other smaller, but nonetheless clear, spectral changes indicate that alterations along the retinal polyene chain also occur. The changes in the retinal structure are preliminary to the deprotonation of the Schiff base nitrogen during the formation of M-412. The time constant for the ps/ns K-590 transformation is estimated from the amplitude change of four vibrational bands in the HOOP region to be 40-70 ns.     

Picosecond time-resolved absorption and fluorescence dynamics in the artificial bacteriorhodopsin pigment BR6.11.

The picosecond molecular dynamics in an artificial bacteriorhodopsin (BR) pigment containing a structurally modified all-trans retinal chromphore with a six-membered ring bridging the C11=C12-C13 positions (BR6.11) are measured by picosecond transient absorption and picosecond time-resolved fluorescence spectroscopy. Time-dependent intensity and spectral changes in absorption in the 570-650-nm region are monitored for delays as long as 5 ns after the 7-ps, 573-nm excitation of BR6.11. Two intermediates, J6.11 and K6.11/1, both with enhanced absorption to the red (> 600 nm) of the BR6.11 spectrum are observed within approximately 50 ps. The J6.11 intermediate decays with a time constant of 12 +/- 3 ps to form K6.11/1. The K6.11/1 intermediate decays with an approximately 100-ps time constant to form a third intermediate, K6.11/2, which is observed through diminished 650-nm absorption (relative to that of K6.11/1). No other transient absorption changes are found during the remainder of the initial 5-ns period of the BR6.11 photoreaction. Fluorescence in the 650-900-nm region is observed from BR6.11, K6.11/1, and K6.11/2, but no emission assignable to J6.11 is found. The BR6.11 fluroescence spectrum has a approximately 725-nm maximum which is blue-shifted by approximately 15 nm relative to that of native BR-570 and is 4.2 +/- 1.5 times larger in intensity (same sample optical density). No differences in the profile of the fluorescence spectra of BR6.11 and the intermediates K6.11/1 and K6.11/2 are observed. Following ground-state depletion of the BR6.11 population, the time-resolved fluroescence intensity monitored at 725 nm increases with two time constants, 12 +/- 3 and approximately 100 ps, both of which correlate well with changes in the picosecond transient absorption data.(ABSTRACT TRUNCATED AT 250 WORDS)     

A time-resolved spectral study of the K and KL intermediates of bacteriorhodopsin.

Nanosecond time-resolved absorption measurements on the photolysis products of bacteriorhodopsin (BR) in intact membranes are reported. At room temperature in fluid solution a single intermediate (KL) is seen 10 ns after excitation. Both spectral and kinetic results are consistent with the KL intermediate converting to the L intermediate by a single first order reaction. The observed temperature-dependent rate has the Arrhenius parameters: Ea = 10.5 kcal/mol, A = 5 x 10(13) s-1. The precursor to the KL intermediate is also observed. Its spectral character is consistent with the K intermediate which has been previously reported. The current data is consistent with a linear sequence in the BR photocycle for K, KL, and L in room temperature fluid solution. Differences in the spectral characteristics of the K intermediates described here and elsewhere are discussed in terms of differences in the microenvironment around the retinal moiety and the affect this may have on the conformation of the chromophore.     

Time-resolved absorption and fluorescence from the bacteriorhodopsin photocycle in the nanosecond time regime

Picosecond transient absorption (PTA) in the 568-660-nm region is measured over the initial 80 ns of the bacteriorhodopsin photocycle. After photocycle initiation with 573-nm excitation (7-ps pulsewidth), these PTA data reflect the formation during the initial 40 ps of two long-recognized intermediates with red-shifted (relative to that of BR-570) absorption bands, namely J-625 and K-590. PTA signals at 568, 628, and 652 nm are unchanged for the remainder of the 80-ns photocycle interval measured, demonstrating that no other intermediates, including the proposed KL, are observable by absorption changes. Picosecond time-resolved fluorescence (PTRF), measured at 740 nm, is initiated by 7 ps excitation of the species present at various time delays after the photocycle begins. PTRF signals change rapidly over the initial 40 ps, reflecting, first, the depletion of the ground state BR-570 population and, subsequently, the formation of K-590. The PTRF signal then decreases monotonically with a time constant of 5.5 ± 0.5 ns from its maximum near a 50-ps delay until it reaches a minimum at a delay of ≈ 13 ns. For time delays between 13 and 80 ns, the PTRF signal remains unchanged and slightly higher than that measured from BR-570 alone. The rapid decrease in PTRF signals over the same photocycle interval in which the PTA signals remain unchanged suggests that the retinal-protein interactions involving electronically excited K-590 (K*) are being significantly altered.     

Azide-binding studies reveal type 3 copper heterogeneity in ascorbate oxidase from the green zucchini squash (Cucurbita pepo).

Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with 'high affinity' (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with 'low affinity' (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.     

Triplet formation on a monomeric chlorophyll in the photosystem II reaction center as studied by time-resolved infrared spectroscopy

The process of formation of the triplet state of chlorophyll in the photosystem II (PS II) reaction center complex was studied by means of time-resolved infrared (IR) spectroscopy. Using a dispersive-type IR spectrometer with a time resolution of approximately 55 ns, transient spectra in the C=O stretching region (1760--1600 cm(-1)) were measured at 77 K. The data were analyzed by singular-value decomposition and subsequent least-squares fitting. Two distinct spectral components having different kinetic behaviors were resolved. One had spectral features characterized by negative peaks at 1740 and 1680 cm(-1) and an overall positive background and was assigned to the P(680)(+)Phe(-)/P(680)Phe radical pair by static FTIR measurements of the P(680)(+)/P(680) and Phe(-)/Phe differences. The other had prominent negative and positive peaks at 1668 and 1628 cm(-1), respectively, which were previously assigned to the keto C==O change upon triplet formation of the monomeric chlorophyll denoted as Chl(T) [Noguchi, T., Tomo, T., and Inoue, Y. (1998) Biochemistry 37, 13614-13625]. The former component of P(680)(+)Phe(-)/P(680)Phe exhibited a multiphasic decay with time constants of 77 ns (75%), 640 ns (18%), 8.3 micros (4%), and 0.3 ms (3%), while the latter component of (3)Chl(T)/Chl(T) was formed with a single-exponential rise with a time constant of 57 ns and had a lifetime of 1.5 ms. From the observations that only the two spectral components were resolved without any other triplet intermediates and the time constant of (3)Chl(T) formation roughly agreed with or seemed even faster than that of the major phase of the P(680)(+)Phe(-) decay, two alternative mechanisms of triplet formation are proposed. (i) (3)Chl(T) is directly formed from P(680)(+)Phe(-) by charge recombination at Chl(T), and (ii) (3)P(680) is formed, and then the triplet is transferred to Chl(T) with a time constant of much less than 50 ns. The location of Chl(T) in the D1 subunit as the monomer chlorophyll corresponding to the accessory bacteriochlorophyll in the L subunit of purple bacteria is favored to explain the former mechanism as well as the triplet properties reported in the literature. The physiological role of the triplet formation on Chl(T) is also discussed.     

The intrinsic tyrosine fluorescence of histone H1. Steady state and fluorescence decay studies reveal heterogeneous emission

In wavelength-resolved steady state spectra we observe three different kinds of emission from histone H1, a class A protein with only a single tyrosine residue. Unfolded H1 emissions that peak at approximately 300 and 340 nm can both be excited maximally at approximately 280 nm. Another, peaking much further to the red at approximately 400 nm, can be excited maximally at approximately 320 nm. The 300-nm fluorescence can be resolved by lifetime measurements into three components with decay times of approximately 1, 2, and 4 ns. On sodium-chloride-induced refolding of H1, simplification of the emission properties occurs. The 340 and 400-nm components disappear while the two shorter lifetime components of the 300-nm band diminish in amplitude and are replaced by the 4-ns decay. We believe that the 340-nm emission is tyrosinate fluorescence resulting from excited-state proton transfer. The origin of the 400-nm emission remains uncertain. We assign the 1 and 2-ns components of the 300-nm emission to two states of tyrosine in denatured H1 and the 4-ns decay to fluorescence of the single tyrosine residue in the globular region of refolded H1. Our results support the contention that salt induced folding of H1 is a cooperative two state process, and permit us to better understand the previously reported increases in fluorescence intensity and anisotropy on salt-induced folding.     

大白鼠糖致白内障的激光喇曼光谱研究

作者用激光喇曼光谱法分析半乳糖导致大白鼠晶状体混浊过程中构象的变化。通过SPEX 1403型激光喇曼光谱仪得到了正常及不同混浊度晶状体的喇曼光谱。结果表明晶状体可溶性蛋白质二级结构的光谱未见异常,其残基酪氨酸及色氨酸微环境起了变化。随着晶状体混浊度的增加,SH谱峰强度变小而S-S键谱峰增强,同时观察到荧光背景逐渐加强。经分析认为晶状体混浊是与蛋白质分子的聚集有关。     

Picosecond dynamics of bacteriorhodopsin, probed by time-resolved infrared spectroscopy.

The photoinduced reaction cycle of bacteriorhodopsin (BR) has been studied by means of a recently developed picosecond infrared spectroscopic method at ambient temperature. BR - K difference spectra between 1560 and 1700 cm-1 have been recorded at delay times from 100 ps to 14 ns. The spectrum remains unchanged during this period. The negative difference OD band at 1660 cm-1 indicates the peptide backbone responds within 50 ps. A survey in the region of carboxylic side chain absorption around 1740 cm-1 reveals that perturbations of those groups, present in low-temperature FTIR spectra, are not observable within 10 ns, suggesting a slow conformational change.     

Low-frequency fourier transform infrared spectroscopy of the oxygen-evolving and quinone acceptor complexes in photosystem II

The low-frequency (<1000 cm-1) region of the IR spectrum has the potential to provide detailed structural and mechanistic insight into the photosystem II/oxygen evolving complex (PSII/OEC). A cluster of four manganese ions forms the core of the OEC and diagnostic manganese-ligand and manganese-substrate modes are expected to occur in the 200-900 cm-1 range. However, water also absorbs IR strongly in this region, which has limited previous Fourier transform infrared (FTIR) spectroscopic studies of the OEC to higher frequencies (>1000 cm-1). We have overcome the technical obstacles that have blocked FTIR access to low-frequency substrate, cofactor, and protein vibrational modes by using partially dehydrated samples, appropriate window materials, a wide-range MCT detector, a novel band-pass filter, and a closely regulated temperature control system. With this design, we studied PSII/OEC samples that were prepared by brief illumination of O2 evolving and Tris-washed preparations at 200 K or by a single saturating laser flash applied to O2 evolving and inhibited samples at 250 K. These protocols allowed us to isolate low-frequency modes that are specific to the QA-/QA and S2/S1 states. The high-frequency FTIR spectra recorded for these samples and parallel EPR experiments confirmed the states accessed by the trapping procedures we used. In the S2/S1 spectrum, we detect positive bands at 631 and 602 cm-1 and negative bands at 850, 679, 664, and 650 cm-1 that are specifically associated with these two S states. The possible origins of these IR bands are discussed. For the low-frequency QA-/QA difference spectrum, several modes can be assigned to ring stretching and bending modes from the neutral and anion radical states of the quinone acceptor. These results provide insight into the PSII/OEC and demonstrate the utility of FTIR techniques in accessing low-frequency modes in proteins.     

Hydrophobic surfaces of tubulin probed by time-resolved and steady-state fluorescence of nile red

Binding of Nile Red to tubulin enhances and blue-shifts fluorescence emission to about 623 nm with a "shoulder" around 665 nm. Binding is reversible and saturable with an apparent Kd of approximately 0.6 microM. Nile Red does not alter tubulin polymerization, and polymerization in 2-(N-morpholino)ethanesulfonic acid (Mes) buffer does not alter the spectrum of the Nile Red-tubulin complex. In contrast, polymerization in glutamate buffer results in a red shift, reduction of intensity, and a decrease in lifetime, suggesting an increase in "polarity" of the binding environment. Lifetimes of 4.5 and 0.6 ns fluorescence in Mes buffer are associated with the 623-nm peak and the 665-nm shoulder, respectively. Indirect excitation spectra for these components are distinct and the 4.5-ns component exhibits tryptophan to Nile Red energy transfer. Acrylamide quenching yields linear Stern-Volmer plots with unchanged lifetimes, indicating static quenching. Apparent quenching constants are wavelength-dependent; global analysis reveals a quenchable component corresponding to the 4.5 ns component and an "unquenchable" component superposing the 0.6-ns spectrum. Analysis of anisotropy decay required an "associative" model which yielded rotational correlation times of greater than 50 ns for the 4.5-ns lifetime and 0.3 ns for the 0.6-ns lifetime. Dilution of tubulin in Mes results in an apparent red shift of emission without lifetime changes, due only to loss of the 623-nm component. These data are reconciled in terms of a model with two binding sites on the tubulin dimer. The more "nonpolar" site is located in a region of subunit-subunit contact which accounts for the fluorescence changes upon dilution; this permits estimation of a subunit dissociation constant of 1 microM.     

Electron paramagnetic resonance of the excited triplet state of metal-free and metal-substituted cytochrome c.

The photoactivated metastable triplate states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters D for the free-base, Zn and Sn derivatives are 465 x 10(-4), 342 x 10(-4) and 353 x 10(-4) cm-1, respectively, and are temperature invariant over the temperature ranges studied. AN E value at 4 K of 73 x 10(-4) cm-1 was obtained for Zn cytochrome c, larger than any previously found for Zn porphyrins derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though the E value for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the T2 > zero-field spin sublevel. EPR line shape changes with respect to temperature of Zn cyt c are interpreted in terms of vibronic coupling, and a maximum Jahn-Teller crystal-field splitting of approximately 180 cm-1 is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is less well resolved in the X-Y region. The magnitude of E value is approximately 60 x 10(-4) cm-1 at 4 K; its value rapidly tends toward zero with increasing temperature, from which a value for the Jahn-Teller crystal-field splitting of > or = 40 cm-1 is estimated. In contrast to those for the metal cytochromes, the magnitude of E value for the free-base derivative was essentially zero at all temperatures studied. This finding is discussed as a consequence of an excited-state tautomerization process that occurs even at 4 K.     

Variation in the Complex Carbohydrate Biosynthesis Loci of Acinetobacter baumannii Genomes

Extracellular polysaccharides are major immunogenic components of the bacterial cell envelope. However, little is known about their biosynthesis in the genus Acinetobacter, which includes A. baumannii, an important nosocomial pathogen. Whether Acinetobacter sp. produce a capsule or a lipopolysaccharide carrying an O antigen or both is not resolved. To explore these issues, genes involved in the synthesis of complex polysaccharides were located in 10 complete A. baumannii genome sequences, and the function of each of their products was predicted via comparison to enzymes with a known function. The absence of a gene encoding a WaaL ligase, required to link the carbohydrate polymer to the lipid A-core oligosaccharide (lipooligosaccharide) forming lipopolysaccharide, suggests that only a capsule is produced. Nine distinct arrangements of a large capsule biosynthesis locus, designated KL1 to KL9, were found in the genomes. Three forms of a second, smaller variable locus, likely to be required for synthesis of the outer core of the lipid A-core moiety, were designated OCL1 to OCL3 and also annotated. Each K locus includes genes for capsule export as well as genes for synthesis of activated sugar precursors, and for glycosyltransfer, glycan modification and oligosaccharide repeat-unit processing. The K loci all include the export genes at one end and genes for synthesis of common sugar precursors at the other, with a highly variable region that includes the remaining genes in between. Five different capsule loci, KL2, KL6, KL7, KL8 and KL9 were detected in multiply antibiotic resistant isolates belonging to global clone 2, and two other loci, KL1 and KL4, in global clone 1. This indicates that this region is being substituted repeatedly in multiply antibiotic resistant isolates from these clones.     

Equilibrium structure and folding of a helix-forming peptide: circular dichroism measurements and replica-exchange molecular dynamics simulations

We have performed experimental measurements and computer simulations of the equilibrium structure and folding of a 21-residue alpha-helical heteropeptide. Far ultraviolet circular dichroism spectroscopy is used to identify the presence of helical structure and to measure the thermal unfolding curve. The observed melting temperature is 296 K, with a folding enthalpy of -11.6 kcal/mol and entropy of -39.6 cal/(mol K). Our simulations involve 45 ns of replica-exchange molecular dynamics of the peptide, using eight replicas at temperatures between 280 and 450 K, and the program CHARMM with a continuum solvent model. In a 30-ns simulation started from a helical structure, conformational equilibrium at all temperatures was reached after 15 ns. This simulation was used to calculate the peptide melting curve, predicting a folding transition with a melting temperature in the 330-350 K range, enthalpy change of -10 kcal/mol, and entropy change of -30 cal/(mol K). The simulation results were also used to analyze the peptide structural fluctuations and the free-energy surface of helix unfolding. In a separate 15-ns replica-exchange molecular dynamics simulation started from the extended structure, the helical conformation was first attained after approximately 2.8 ns, and equilibrium was reached after 10 ns of simulation. These results showed a sequential folding process with a systematic increase in the number of hydrogen bonds until the helical state is reached, and confirmed that the alpha-helical state is the global free-energy minimum for the peptide at low temperatures.     

Dynamics of the excited state of the primary electron donor in reaction centers of Rhodopseudomonas viridis as revealed by hole burning at 1.7K. Different conformational states

The spectra of absorbance changes (delta A) due to the formation of P+Q- (P, primary electron donor, Q, primary quinone acceptor) at 1.7K in Rhodopseudomonas viridis reaction centers (RCs) excited at 1014 nm has been shown to include, besides a progression of broad (170-190 cm-1) Gaussian vibronic bands separated by 150 cm-1, a 'narrow' structure near 1014 nm which can be simulated by a Lorentian zero-phonon hole (ZPH) and Lorentian one-mode (26.8 cm-1) phonon wings. The widths of ZPH of approximately 17 cm-1 for delta A reflecting the formation of P+Q- decaying in the ms time domain and of 6.8 +/- 0.4 cm-1 for P+Q- decaying in the min time domain at 1.7K, seems to correspond to different conformations of RCs with a relaxation time of P* of approximately 0.6 ps (in agreement with measurements in this time domain) and 1.6 +/- 0.1 ps, respectively. The comparison of the spectra of delta A in the region of the BL band for slow (min) and fast (ms) decaying components suggests a different mutual arrangement of P and BL for different conformations of RCs. It is assumed that the broad and narrow structures of the P band reflect the transitions to two configurations with different P-protein interactions. 'Narrow' structure of delta A spectrum with essentially the same phonon wings and ZPH (width of 3.8 +/- 0.4 cm-1) was observed within the P band when HL was photoreduced at 1.7K.     

Time-resolved detection of energy transfer: theory and application to immunoassays

Energy-transfer measurements based upon acceptor fluorophore emission are plagued with background fluorescence resulting from absorption of the excitation light by the acceptor fluorophore. The present work examines the use of a long-lifetime donor fluorophore and a short-lifetime acceptor fluorophore, combined with pulsed-laser excitation and electronic gating of detector signals, to separate the component of acceptor emission due to energy transfer from the component due to absorption of the excitation light. Theoretical equations describing the acceptor fluorescence and integrated acceptor fluorescence show that increasing the integration delay relative to the excitation pulse should greatly enhance detection of the energy-transfer component. The time-resolved detection of energy transfer was tested in a competitive immunoassay format in which antibodies to human immunoglobulin G (IgG) F(ab')2 fragments were covalently labeled with pyrenebutyrate (tau = 100 ns) and IgG Fab' fragments were covalently labeled with B-phycoerythrin (tau = 2.5 ns). Solutions containing these two conjugates exhibited energy transfer from the pyrenebutyrate to the B-phycoerythrin upon excitation with a nitrogen laser. Acceptor emission was measured with 0- and 20-ns integration delays and the ratios of the energy-transfer component to the laser-excited component were found to increase by 9- to 15-fold when the 20-ns delay was used in three series of immunoassays. Good agreement between the experimental data and theory was obtained following convolution of the theoretical fluorescence responses with the instrumental response of the fluorometer.     

Temperature jump-induced secondary structural change of the membrane protein bacteriorhodopsin in the premelting temperature region: a nanosecond time-resolved Fourier transform infrared study

The secondary structural changes of the membrane protein, bacteriorhodopsin, are studied during the premelting reversible transition by using laser-induced temperature jump technique and nanosecond time-resolved Fourier transform infrared spectroscopy. The helical structural changes are triggered by using a 15 degrees C temperature jump induced from a preheated bacteriorhodopsin in D2O solution at a temperature of 72 degrees C. The structural transition from alphaII- to alphaI-helices is observed by following the change in the frequency of the amide I band from 1667 to 1651 cm-1 and the shift in the frequency of the amide II vibration from 1542 cm-1 to 1436 cm-1 upon H/D exchange. It is found that although the amide I band changes its frequency on a time scale of <100 ns, the H/D exchange shifts the frequency of the amide II band and causes a complex changes in the 1651-1600 cm-1 and 1530-1430 cm-1 frequency region on a longer time scale (>300 ns). Our result suggests that in this "premelting transition" temperature region of bacteriorhodopsin, an intrahelical conformation conversion of the alphaII to alphaI leads to the exposure of the hydrophobic region of the protein to the aqueous medium.     

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

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

Urine analysis using FTIR spectroscopy: A study on healthy adults and children

Urine spectra from 108 healthy volunteers are studied by attenuated total refraction-Fourier transform infrared (ATR-FTIR) spectroscopy. The spectral features are correlated with observable urine components. The variation of spectra within a healthy population is quantified and a library of reference spectra is constructed. Using the band assignments, these spectra are compared with both age-wise and gender-wise. Children show the least intensity variations compared to both adult groups. Young adults show the highest variation, particularly in the 1650 to 1400 cm⁻¹ and 1200 to 900 cm⁻¹ regions. These results indicate the importance of the size of the control group in comparative studies utilizing FTIR. Age-wise comparisons reveal that phosphate and sulfate excretion decreases with age, and that the variance of phosphate among individuals is higher with adults. As for gender-wise comparisons, females show a slightly higher citrate content at 1390 cm⁻¹ regardless of the age and they show a higher variance in the 1200 to 1000 cm⁻¹ region when compared to men.     

Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures

Picosecond laser spectroscopic analysis was applied to determine how many intermediates existed in the primary photochemical process of trans-bacteriorhodopsin (light-adapted bacteriorhodopsin) at room temperature (18°C) and to calculate their absorption spectra. Irradiation of bacteriorhodopsin with a laser pulse (wavelength, 532 nm; pulse width, 25 ps) yielded the K intermediate (K) which was produced through a precursor, having an absorption maximum (λ_max) longer than that of K. K was stable during a picosecond time range (50–900 ps). The λ_max was located at 610 nm and the extinction coefficient (?_max) was 0.92-times that of bacteriorhodopsin. The same K intermediate was produced from bacteriorhodopsin even when it was excited with a high-energy pulse by which a saturation effect was induced. A transient difference spectrum measured at 150 ns after the excitation of bacteriorhodopsin was different in shape from that of the K intermediate, suggesting that an intermediate was formed by thermal decay of K. This intermediate, tentatively called the KL intermediate (KL), had a λ_max at 596 nm and an ?_max 0.80-times that of bacteriorhodopsin. KL decayed to the L intermediate (L) with a time constant of 2.2 μs. L has a λ_max at 543 nm and an ?_max 0.66-times that of bacteriorhodopsin.