Vibrational spectroscopy of an algal Phot-LOV1 domain probes the molecular changes associated with blue-light reception

The LOV1 domain of the blue light Phot1-receptor (phototropin homolog) from Chlamydomonas reinhardtii has been studied by vibrational spectroscopy. The FMN modes of the dark state of LOV1 were identified by preresonance Raman spectroscopy and assigned to molecular vibrations. By comparing the blue-light-induced FTIR difference spectrum with the preresonance Raman spectrum, most of the differences are due to FMN modes. Thus, we exclude large backbone changes of the protein that might occur during the phototransformation of the dark state LOV1-447 into the putative signaling state LOV1-390. Still, the presence of smaller amide difference bands cannot be excluded but may be masked by overlapping FMN modes. The band at 2567 cm(-1) is assigned to the S-H stretching vibration of C57, the residue that forms the transient thio-adduct with the chromophore FMN. The occurrence of this band is evidence that C57 is protonated in the dark state of LOV1. This result challenges conclusions from the homologous LOV2 domain from oat that the thiolate of the corresponding cysteine is the reactive species.     

Fourier transform IR spectroscopy study for new insights into molecular properties and activation mechanisms of visual pigment rhodopsin

Fourier transform IR (FTIR) spectroscopy has been successfully applied in recent years to examine the functional and structural properties of the membrane protein rhodopsin, a prototype G protein coupled receptor. Unlike UV-visible spectroscopy, FTIR spectroscopy is structurally sensitive. It may give us both global information about the conformation of the protein and very detailed information about the retinal chromophore and all other functional groups, even when these are not directly related to the chromophore. Furthermore, it can be successfully applied to the photointermediates of rhodopsin, including the active receptor species, metarhodopsin II, and its decay products, which is not expected presently or even in the near future from crystallographic approaches. In this review we show how FTIR spectroscopy has significantly contributed to the understanding of very different aspects of rhodopsin, comprising both structural properties and the mechanisms leading to receptor activation and deactivation.     

Spectroimmunochemistry using colloidal gold bioconjugates

Using surface-enhanced infrared absorption (SEIRA) spectroscopy of dry films of colloidal gold (CG) bioconjugates with protein A, it is shown that certain characteristic bands of the protein (e.g., amide I, amide II and some other vibration modes) are essentially affected by the metal surface. Thus, the method may be used for controlling the quality of such bioconjugates. Moreover, it is demonstrated that the biospecific reaction of protein A attached to CG particles with human immunoglobulin G (IgG) results in further essential changes in SEIRA spectra, providing a means for an easy and rapid IR spectroscopic detection of biospecific immunochemical interactions (i.e., spectroimmunochemistry). The results obtained can form a basis for developing test systems for detecting various biospecific interactions.     

Folding,stability, and secondary structure of a new dimeric cysteine proteinase inhibitor

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high beta-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical beta-structure, but similar to those of a group of small beta-strand proteins, with a peak at 189nm and a trough at 202nm. An aromatic peak at 232nm and infrared bands at 1633 and 1515cm(-1) associated with the peptide backbone and the tyrosine microenvironment, respectively, were used to characterize the thermal unfolding. The reversible transition has a midpoint at 67 degrees C, with DeltaG=34kJ/mol and DeltaH=300kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44kJ/mol, respectively. A model for folding and assembly is discussed.     

Fourier transform IR study of aggregational behavior of N-acetyl-L- and N-butyloxycarbonyl-L-glutamic acid oligomeric benzyl esters in dioxane and benzene: beta-turn --> antiparallel beta-sheet transition

Resins from several different genera are studied using Fourier transform (FT)-Raman spectroscopy. Tree resins can be broadly divided into those that contain diterpenoid components and those that contain triterpenoid components. The diterpenoid resins analyzed are from the genera Pinus, Cedrus, and Agathis (kauri resin) and the triterpenoid resins examined are samples from Pistacia, Boswellia (frankincense), and Commiphora (myrrh) genera. A protocol is developed to nondestructively distinguish diterpenoid and triterpenoid resins and to differentiate the genera within the two types. The effects of oxidation on the discrimination of the FT-Raman spectra are considered.     

Gamma irradiation and cellular damage in Kocuria rosea: investigation by one- and two-dimensional infrared spectroscopy

Fourier transform infrared (FT-IR) spectroscopy was used to investigate the radiation-induced effects on Kocuria rosea. Bacterial suspensions at the stationary phase were exposed to increasing doses of gamma radiation. In the region 1350-840cm(-1), assigned to phosphodiester backbone, nucleic acids, and sugar rings, the radical damaging effects were dose-dependent, with the first threshold at 2.75kGy and the second at 13.75kGy inducing more striking spectral variations. Postirradiation reincubation did not significantly affect the biomolecular response, except in the spectral range 1100-1000cm(-1). These observations suggest the occurrence of new phylogenetic characteristics for K. rosea following irradiation. Moreover, two-dimensional analysis was used to highlight correlated evolutions of molecular species as radical aggression increased. The results point to an evolutionary scheme during the time course of irradiation. Thus, one- and two-dimensional IR analyses are convenient means of investigating the metabolic events following oxidative stress generated by either chemical or physical agents.     

Orientation selection in photosynthetic PS I multilayers: structural investigation of the charge separated state P700A1 by high-field/high-frequency time-resolved EPR at 3.4 T/95 GHz

The radical-pair state of the primary electron donor and the secondary electron acceptor (P₇₀₀⁺A₁⁻) of the photosynthetic reaction center (RC) photosystem I (PS I) of Synechocystis PCC 6803 was studied by time-resolved electron paramagnetic resonance (TREPR) at high field/high frequency (3.4 T/95 GHz) using orientation selection in multilayers. The goal of the present article is to work out the basis for future studies, in which the improved resolution of such multilayers may be used to detect mutation-induced structural changes of PS I in membrane preparations. This approach is particularly interesting for systems that cannot be prepared as single crystals. However, in order to use such multilayers for structural investigations of protein complexes, it is necessary to know their orientation distribution. PS I was chosen as a test example because the wild type was recently crystallized and its X-ray structure determined to 2.5 Å resolution [Nature 411 (2001) 909]. On the basis of our experimental results we determined the orientation distribution. Furthermore, a simulation model for the general case in which the orientation distribution is not axially symmetric about the C₂ symmetry axis of the RC is developed and discussed. Spectra simulations show that changes in the TREPR spectra of PS I are much more significant for these oriented multilayers than for disordered samples. In this way the use of oriented multilayers, in conjunction with multifrequency TREPR measurements on oriented as well as on disordered samples, is a promising approach for studies of structural changes of PS I systems that are induced by point mutations.     

Simultaneous and rapid monitoring of biomass and biopolymer production by Sphingomonas paucimobilis using Fourier transform-near infrared spectroscopy

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.     

Statistical and Frequency-Amplitude Characteristics of Ultraweak Emissions of the Loach Eggs and Embryos under the Normal Conditions and upon Their Optic Interactions. 2. Changes in Characteristics of Ultraweak Emissions upon Optic Interaction of Groups of Embryos of Different Ages

We compared the characteristics of ultraweak emissions from groups of loach embryos of different ages in the presence or absence of optic interaction. The percentage of zero values of emission gradually increased during the first hour of optic interaction. The number and height of rare big pulses estimated by the value of kurtosis increased in parallel. In addition, the correlation between the Fourier spectra of optically interacting samples decreased at a higher rate than in the absence of optical contact. Just after the 1-hour optic interaction was terminated, the number of high pulses decreased in a younger interacting group and increased in the older one and the farther away the partner groups were in developmental stages, the more pronounced these differences were. Measurements of the Fourier spectra after long-term (12–22-hour) optic interactions have shown that an exchange of autocorrelation characteristics of the spectra took place among the samples: the sums of autocorrelation coefficients were inverted in the vast majority of cases, often with an overshoot or, at least, were smoothed over with reference to the control samples. We conclude that the previously described effects of optic interactions between groups of loach embryos of different ages could be due to changes in the frequency spectra of their ultraweak emissions.     

A biomechanical and spectroscopic study of bone from rats with selenium deficiency and toxicity

Selenium, being an essential mineral in the mammalian diet, is important in providing protection against oxidative damage. Numerous in vitro studies of selenium compounds reveal a very high correlation between catalytic activity of selenium compounds and toxicity. The present study was designed to investigate the effects of dietary selenium on the biomechanical properties of bone. New born rats of both sexes were fed with either a control, or a selenium- and vitamin E-deficient, or a selenium-excess and vitamin E-adequate diet. We obtained the stiffness (modulus of elasticity) of bones (femur and tibia) by tensile test for all groups considered. Both the deficient and the excess groups have decreased biomechanical strength with respect to the control group. To support our biomechanical results for both experimental groups, X-ray diffraction analysis and FTIR spectroscopic study were performed on the femurs and tibiae. The X-ray diffraction analysis showed that the intensities of the peak observed at around 2°=31.820, in the control femur and tibia are stronger than the intensities of the corresponding peak of two experimental groups. In FTIR spectroscopy, the disappearance and/or reduction of the intensities of some carbonate bands in the two experimental groups indicate that there is a decrease in crystallinity and mineral contents which, together with X-ray diffraction analysis, correlate very well with the biomechanical data.