Jamal F.M. Almarashi, Natalia Kapel, Thomas S. Wilkinson, Helmut H. Telle
205 - 208
Rapid and reproducible discrimination between bacterial pathogens is a clear goal in microbiological laboratories when processing infected clinical samples. In this study Raman spectra were taken from at least 30 colonies of four strains of bacteria including Staphylococus epidermidis (1457 and 9142) and Escherichia coli (K12 and Top10) using the Renishaw inVia Raman microscope system. Analysis based on principal components suggests that even strain differentiation (e.g., 1457 vs. 9142 or K12 vs Top10) is possible.
Andreas A. Deeg, Tobias E. Schrader, Halina Strzalka, Jose Pfizer, Luis Moroder, Wolfgang Zinth
209 - 213
The light driven disassembly process of amyloid-like structures formed by azobenzene model peptides is studied by time resolved mid-IR spectroscopy from nanoseconds to minutes. The investigated peptide consists of two amino acid strands connected by the azobenzene switch. The peptides aggregate to amyloid-like structures when the azobenzene chromophore is in the trans conformation. Illumination, resulting in a trans to cis isomerization of the azobenzene, leads to disaggregation of the aggregated structures. After optical excitation and isomerization of the azobenzene one finds absorption changes which recover to a large extent on the time scale of few nanoseconds. These early absorption transients are assigned to the relaxation of vibrational excess energy (heat) or to structural rearrangements of isomerized azobenzene and the aggregated surroundings. It is only on the timescale of minutes that spectral signatures appear which are characteristic for the disassembly of the aggregated structure.
M. Polakovs, N. Mironova-Ulmane, A. Pavlenko, E. Reinholds, M. Gavare, M. Grube
215 - 219
In the present work we report results of investigations of human blood before and after radioisotope Tc99m diagnosis by Electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopies. It is shown, that EPR can detect the concentration of methaemoglobin and transferrin ions of more accurately than any other technique. FTIR spectra indicated radiation caused conformational and concentration changes of proteins. Hierarchical cluster analysis (HCA) was created as time-saving tool for discrimination of initial and irradiated in vivo human blood samples.
V. Parfejevs, M. Gavare, L. Cappiello, M. Grube, R. Muceniece, U. Riekstina
221 - 225
Recently FT-IR analysis has been employed to study changes in molecular signatures during embryonic stem cell differentiation. We were interested to find out whether FT-IR spectroscopy could be applied to analyze changes in human skin mesenchymal stem cell (S-MSC) biochemical profile during in vitro neurodifferentiation.
S-MSCs were propagated in serum-free medium with EGF and FGF-2 during six weeks. Neural progenitor cell line ReNcell CX (Millipore) was used as a reference cell line. Samples were collected each week and analyzed for neural marker nestin, tubulin βIII, GFAP and CD271 expression. FT-IR analysis was carried out using microplate reader HTS-XT (Bruker, Germany).
Despite the immunophenotype similarity, FT-IR spectroscopy revealed distinct profiles for S-MSC culture and ReNcell CX cells. FT-IR spectra analyses showed changes of protein and lipid concentration during neurodifferentiation and different carbohydrate composition in ReNcell CX and S-MSCs. It was possible to discriminate between S-MSC cultures at different time-points during neurodifferentiation.
The results of this study demonstrate that FT-IR spectroscopy is more sensitive than conventional immunophenotyping analysis and it has a great potential for the monitoring of the stem cell differentiation status.
E. Regulska, M. Samsonowicz, R. Świsłocka, W. Lewandowski
227 - 233
Optimized geometrical structures of alkali metal phenoxyacetates were obtained using B3LYP/6-311++G** method. Geometric and magnetic aromaticity indices, dipole moments and energies were calculated. Atomic charges on the atoms of phenoxyacetic acid molecule and its alkali metal salts were calculated by Mulliken, APT (Atomic Polar Tensor), NPA (Natural Population Analysis), MK (Merz–Singh–Kollman method) and ChelpG (Charges from electrostatic potentials using Grid based method) methods. The theoretical wavenumbers and intensities of IR as well as chemical shifts in NMR spectra were obtained and compared with experimental data. The effect of alkali metals on molecular structure of phenoxyacetic acid expresses in the shift of selected bands along the series of alkali metal salts. The correlation between chosen bands and some metal parameters, such as electronegativity, ionization energy, atomic and ionic radius have been noticed.
FT-IR, Raman and NMR spectra of phenoxyacetic acid and its sodium salt were recorded and analyzed. Optimized geometrical structures of studied compounds were calculated by B3LYP/6-311++G** method. The atomic charges were calculated by Mulliken, NPA (Natural Population Analysis), APT (Atomic Polar Tensor), MK (Merz–Singh–Kollman method) and ChelpG (Charges from electrostatic potentials using Grid based method) methods. Geometric as well as magnetic aromaticity indices, dipole moments and energies were also calculated. The theoretical wavenumbers and intensities of IR spectra as well as chemical shifts in 1H and 13C NMR spectra were obtained. The calculated parameters were compared with experimental characteristics of these molecules.
Nikolaos Kourkoumelis, Alexandros Polymeros, Margaret Tzaphlidou
243 - 247
Raman spectroscopy grows into an essential tool for biomedical applications. Nevertheless, the weak Raman signal associated mainly with biological samples, is often obscured by a broad background signal due to the intrinsic fluorescence of the organic molecules present, making further analysis unfeasible. A novel computational geometry method based on the definition of convex hull is described to estimate the background from Raman spectra of samples with biological interest. The method is semi-automated requiring sample-dependent user intervention. It does not rely however on curve fitting, requires no information about background distribution or source and keeps the original spectral data intact.
Olesya V. Stepanenko, Olga V. Stepanenko, Irina M. Kuznetsova, Vladislav V. Verkhusha, Konstantin K. Turoverov
249 - 254
The guanidine thiocyanate induced denaturation-renaturation of sfGFP was studied. It was shown that the disruption of sfGFP native structure occurs in the range of guanidine thiocyanate concentrations from 0.5 to 2.5 M. This process was accompanied by simultaneous changes of all recorded parameters. It was found that the small guanidine thiocyanate concentrations (less then 0.1–0.2 M) triggered local structural disturbances in protein which result in significant decrease of chromophore and tryptophan fluorescence intensity and change of protein visible absorption spectrum.
Olga V. Stepanenko, Olesya V. Stepanenko, Alexander V. Fonin, Vladislav V. Verkhusha, Irina M. Kuznetsova, Konstantin K. Turoverov
255 - 260
In this work we have studied peculiarities of protein–ligand interaction under different conditions. We have shown that guanidine hydrochloride (GdnHCl) unfolding–refolding of GGBP in the presence of glucose (Glc) is reversible, but the equilibrium curves of complex refolding-unfolding have been attained only after 10 days incubation of GGBP/Glc in the presence of GdnHCl. This effect has not been revealed at heat-induced GGBP/Glc denaturation. Slow equilibration between the native protein in GGBP/Glc complex and the unfolded state of protein in the GdnHCl presence is connected with increased viscosity of solution at moderate and high GdnHCl concentrations which interferes with diffusion of glucose molecules. Thus, the limiting step of the unfolding–refolding process of the complex GGBP/Glc is the disruption/tuning of the configuration fit between the protein in the native state and the ligand.
The studies about the interaction of actin with vanadium are seldom. In the present report the effects of vanadyl, vanadate and decavanadate in the actin structure and function were compared. Decavanadate clearly interacts with actin, as shown by 51V-NMR spectroscopy. Decavanadate interaction with actin induces protein cysteine oxidation and vanadyl formation, being both prevented by the natural ligand of the protein, ATP. Monomeric actin (G-actin) titration with vanadyl, as analysed by EPR spectroscopy, indicates a 1:1 binding stoichiometry and a kd of 7.5 μM. Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC50 of 68 and 300 μM, respectively, as analysed by light scattering assays. However, only vanadyl induces G-actin intrinsic fluorescence quenching, which suggest the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Finally, both vanadium species increased ε-ATP exchange rate (k=6.5×10−3 and 4.47×10−3 s−1 for decavanadate and vanadyl, respectively). Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl.
Catarina S.H. Jesus, Daniela C. Vaz, Maria J.M. Saraiva, Rui M.M. Brito
267 - 271
Transthyretin (TTR) is a homotetrameric protein implicated in several amyloid diseases. The mechanism by which TTR is converted into elongated fibrillar assemblies has been extensively investigated, and numerous studies showed that dissociation of the native tetrameric structure into partially unfolded monomeric species precedes amyloid formation. The small differences observed in the crystal structures of different TTR variants, as well as the thermodynamics and kinetics of tetramer dissociation, do not seem to completely justify the amyloidogenic potential of different variants. With this in mind, we have studied the refolding kinetics of WT-TTR and its most common amyloidogenic variant V30M-TTR, monitoring changes in intrinsic tryptophan fluorescence at different urea and protein concentrations. Our results demonstrate that the in vitro refolding mechanisms of WT- and V30M-TTR are similar, involving a dimeric intermediate. However, there are large differences in the refolding rate constants for the two variants, specially at nearly native conditions. Interestingly, tetramer formation occurs at a much slower rate in the amyloidogenic variant V30M-TTR than in WT-TTR, which in the in vivo setting may promote the accumulation of monomeric species in the extracellular environment, resulting in higher susceptibility for aggregation and amyloid formation instead of spontaneous refolding.
The activity of enzymes in organic solvents substantially increases in the presence of crown ethers. Tris(hydroxy- methyl)aminomethane (tris) is chosen as a model compound to simulate the interaction of surface amino groups of protein with crown ether. The methods of FTIR and time-domain THz spectroscopy are used to study the interaction of tris with 18-crown-6. The THz spectra of the complexes are measured for the first time.
Barbora Řezáčová, Yves-Marie Coïc, Christian Zentz, Pierre-Yves Turpin, Josef Štěpánek
277 - 282
We have introduced a new promising approach for the determination of pKa constants of oligopeptide intrinsic fluorophores and spectral components referring to their differently charged states. The method is based on the factor analysis of multiwavelength spectroscopic pH titration data. As an illustration we present its application on the study of short segments of the MADS box, which is a highly conserved sequence of a so-called family of transcription factors, by techniques of UV absorption and fluorescence spectroscopies. Investigated oligopeptides contain no tryptophan but one tyrosine serving as an intrinsic fluorophore and absorber. The results indicate both good sensitivity and spectroscopic selectivity of our method, which thus may be considered as a complementary technique to conventional electrochemical methods.
Emilie Bulard, Marie-Pierre Fontaine-Aupart, Henri Dubost, Wanquan Zheng, Jean-Marie Herry, Marie-Noëlle Bellon-Fontaine, Romain Briandet, Bernard Bourguignon
283 - 290
In biomedical and food industry, surface colonization by bacteria is harmful: it leads to biofilm formation, a microbial consortia more resistant to antibiotics than planktonic bacteria. In order to design materials able to limit the biofilm formation, the effect of bacteria on materials has to be well characterized. In this work, a well-defined surface composed of a self-assembled monolayer (SAM) of OctaDecaneThiol (ODT) onto a gold surface is probed in situ. The SAM conformation is obtained using the femtosecond vibrational sum frequency generation (SFG) spectroscopy. This technique provides selectively the molecular vibrational signature of the interface. The behaviour of the ODT SAM is studied in different environments: in air, in water and upon exposure to hydrophilic or hydrophobic Lactococcus lactis bacteria. Modelling the experimental SFG spectra reveals a measurable change of the SAM conformation which depends on the environment, especially on the hydrophilic-hydrophobic character.
M. Grube, R. Rutkis, M. Gavare, Z. Lasa, I. Strazdina, N. Galinina, U. Kalnenieks
291 - 295
Z. mobilis ATCC 29191 and its respiratory knock-out mutants – kat-, ndh-, cytB- and cydB-, were grown under anaerobic and aerobic conditions. FT-IR spectroscopy was used to study the variations of the cell macromolecular composition. Quantitative analysis showed that the concentration ratios – nucleic acids to lipids, for Z. mobilis parent strain, kat-, ndh-, cytB- and cydB- strains, clearly distinguished Z. mobilis parent strain from its mutant derivatives, and corresponded fairly well to the expected degree of biochemical similarity between the strains.
Two different FT-IR-spectra hierarchical cluster analysis (HCA) methods were created to differentiate Z. mobilis parent strain and respiratory knock-out mutant strains. HCA based on discriminative spectra ranges of carbohydrates, nucleic acids and lipids allowed to evaluate the influence of growth environment (aeration, growth phase) on the macromolecular composition of cells and differentiate the strains. HCA based on IR spectra of inoculums, in a diagnostic region including the characteristic nucleic acid vibration modes, clearly discriminated the strains under study. Thus it was shown that FT-IR spectroscopy can distinguish various alterations of Z. mobilis respiratory metabolism by HCA of biomass spectra.