Spectroscopy - Volume 22, issue 4

Authors: Ward, Priscilla | Huang, Chengdong | Banerjee, Monimoy | Mohanty, Smita

Article Type: Research Article

Abstract: Glutaminase interacting protein (GIP), a PDZ domain containing protein, mediates the distribution and clustering of proteins/peptides in membranes, acting as a scaffold where signaling molecules are linked to a multi-protein complex. GIP has been shown to play a key role in the glutamate signaling system. Some metals, particularly Pb2+ , Cu2+ and Zn2+ , have been implicated in a wide range of neurological disorders including Alzheimer's disease and Parkinson's disease, whose etiologies have been associated with dysfunction of the glutamate signaling system. Here, for the first time, the effects of lead, copper, and zinc on GIP were determined by …using circular dichroism and fluorescence spectroscopy. All three metal ions significantly affected the conformational properties of GIP. The deconvolution of CD data showed that, with increasing amounts of Pb2+ /Cu2+ /Zn2+ , the α-helix percentage decreased while the β-sheet content increased. The binding constants of GIP to Pb2+ , Cu2+ and Zn2+ determined by fluorescence were found to be 1.4, 2.38 and 2.85 μM respectively, which indicated strong bindings between GIP and all three metal ions. We propose that the metal ion binding site of GIP is located on α-2 helix, where residues His90, Asp91 and Arg94 may coordinate the metal ions. The conformational change of GIP upon metal ion binding possibly results from the disruption of a salt bridge between Asp91 and Arg94. Show more

Keywords: GIP, PDZ domain, brain diseases, metal ions interaction, fluorescence, CD

DOI: 10.3233/SPE-2008-0345

Citation: Spectroscopy, vol. 22, no. 4, pp. 213-221, 2008

Authors: Aamouche, Ahmed | Goormaghtigh, Erik

Article Type: Research Article

Abstract: We report a new generic device suitable for the investigation of biological interactions by means of Fourier Transform Infrared (FTIR) spectroscopy. The research is focused on multi reflected evanescent optical radiation through a chemically modified surface of the attenuated total reflection (ATR) element (silicon or germanium). Using a wet chemistry approach, the original method is based on grafting of a bifunctional binding molecule (N-hydroxysuccinimidyl ester forms) at the surface of the Si or the Ge crystals. The functionalized surface permits then the foundation of different types of self-assembled phospholipid membranes. The obtained sensors allow the detection, in infrared spectral domain, …of any perceptible molecular interaction or structural changes. The key experimental result concerns the coagulation factor VIII (FVIII ). The principle of the diagnostic is related to the ability of FVIII molecules to bind specifically to phosphatidylserine (PS) membrane. Show more

Keywords: Biosensors, self-assembled lipid membranes, FTIR-ATR spectroscopy, ligand–receptor binding, protein–lipid interactions, factor VIII, haemophilia

DOI: 10.3233/SPE-2008-0351

Citation: Spectroscopy, vol. 22, no. 4, pp. 223-234, 2008

Authors: Mezzetti, Alberto | Spezia, Riccardo

Article Type: Research Article

Abstract: We have used time-resolved step-scan FTIR spectroscopy to investigate the process of triplet formation in the peridinin–chlorophyll-a–protein (PCP) at 100 K. Results have led to the identification of possible marker bands for peridinin (Per) and 3 Per. Band assignment has been carried out performing a DFT investigation on Per, 3 Per and on model molecules. The main spectral feature is a couple of bands at 1746 (negative) and 1719 (positive) cm−1 . The assignment of these two bands to the lactonic C=O of Per in (respectively) its fundamental S0 and triplet T1 state is based on: (1) comparison …with FTIR spectra of isolated Per in literature; (2) good agreement with DFT calculations for Per and 3 Per; (3) the downshift of the band upon triplet formation as predicted by DFT calculations in Per and similar lactonic systems; (4) the kinetic of band disappearance, in agreement with literature data. Another possible band for Per in its fundamental state has been identified at 1523 cm−1 . The results are in overall agreement with a recent step-scan FTIR study on PCP at 298 K (Alexandre et al., Biophys. J. 93 (2007), 2118–2128) even though some small discrepancies emerge, probably related to the different temperature at which experiments were carried out. Further DFT calculations have been performed to rationalise these spectral discrepancies. From a methodological point of view, the work demonstrates the potential of a combined step-scan FTIR/DFT approach in the study of photophysical processes in proteins. Furthermore, it has been shown that, when small differential IR signals are concerned, nagging photothermal effects can interfere and can possibly lead to misleading interpretations. Show more

Keywords: Step-scan FTIR, DFT, triplet state, peridinin, peridinin–chlorophyll-a–protein

DOI: 10.3233/SPE-2008-0355

Citation: Spectroscopy, vol. 22, no. 4, pp. 235-250, 2008

Authors: Porumb, H.

Article Type: Research Article

Abstract: The expression of AML1-ETO, resulting from the t(8; 21) chromosomal translocation causes 15% of acute myeloid leukaemias. The NHR2 region of ETO, bearing the motif LxxLL, is involved in the oligomerisation of the AML1-ETO. “Peptide NHR2” is one of the objects of the present investigation. The TAFH region of ETO may recruit AML1-ETO to transcription activators, such as E-protein. “Peptide TAFH” is another object of the present investigation. TAFH interacts with E-protein through the AD1 domain of the latter, which possesses an LxxLL motif as well. “Peptide AD1” is the third object of the present investigation. By CD, ANS fluorescence …and intrinsic fluorescence, we suggest an antiparallel coiled-coil encounter of two NHR2 molecules (Kd =2.8–4 μM) as a prerequisite to tetramer formation. On the other hand, we show that the TAFH domain would probably recognize another partner bearing the LxxLL motif and, before binding to AD1 (Kd =28 nM), the first such interaction is likely to be intramolecular, with the NHR2 domain of the AML1-ETO protein itself (Kd =1.28 nM). Furthermore, a possible interaction of NHR2 with AD1 is also revealed (Kd =240 nM). The biological implications of the results are discussed. Show more

Keywords: Activity modulation, amphipathic peptides, ANS, binding constants, circular dichroism, coiled coil, intrinsic fluorescence, Newton–Raphson method, oligomerisation

DOI: 10.3233/SPE-2008-0346

Citation: Spectroscopy, vol. 22, no. 4, pp. 251-260, 2008

Authors: Boutin, Céline | Roche, Yann | Millot, Christine | Deturche, Régis | Plain, Jérôme | Jeannesson, Pierre | Manfait, Michel | Royer, Pascal | Millot, Jean-Marc | Jaffiol, Rodolphe

Article Type: Research Article

Abstract: Fluorescence correlation spectroscopy (FCS) has been used to analyze the plasma membrane fluidity and heterogeneity of multidrug resistant cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been explored. Firstly, we reveal a modification of plasma membrane heterogeneities according to the addition of a fluidity modulator, benzyl alcohol. On the other hand, revertants such as verapamil and cyclosporin-A appear to act more specifically on the slow diffusion sites, such as lipids microdomains.

Keywords: Fluorescence correlation spectroscopy, membrane dynamic, multidrug resistant (MDR) cells

DOI: 10.3233/SPE-2008-0348

Citation: Spectroscopy, vol. 22, no. 4, pp. 261-265, 2008

Authors: Vachoušek, Jan | Štěpánek, Josef

Article Type: Research Article

Abstract: Structural characteristics of a central segment of the HIV RNA trans-activation response element (TAR) have been studied in aqueous solution. The investigated segment (TAR-14), comprising nucleotides 26–39 of the wild-type HIV genome (GAGC-CUGGGA-GCUC sequence), forms the TAR apical hairpin with a six-base loop and a stem of 4 base pairs. Raman spectra were recorded in a wide temperature range and the spectral series analyzed by means of factor analysis (FA) and a consequent fit to a thermodynamic model. The model was applied for temperatures above 20°C because of spectroscopic indication of possible intermolecular complexes at lower temperatures. FA results corresponded …to a monomolecular reaction of the hairpin opening characterized by a melting temperature of 71°C and ΔG37 =−3.2 kcal/mol. Besides the process of the hairpin opening, the FA revealed also substantial, approximately linear, temperature induced changes of the closed hairpin Raman spectrum. Spectral changes attributed to this effect indicated partial disordering of the A-type duplex geometry of the stem, opening of intraloop hydrogen bonds, weakened conformational regularity of the loop guanosines and release of the loop uracil. Show more

Keywords: HIV, TAR, RNA, hairpin, Raman spectroscopy, factor analysis

DOI: 10.3233/SPE-2008-0349

Citation: Spectroscopy, vol. 22, no. 4, pp. 267-277, 2008

Authors: Torreggiani, Armida

Article Type: Research Article

Abstract: The damages induced by reductive radical stress on bovine pancreatic ribonuclease A (RNAse A) were investigated by Raman spectroscopy. Gamma-irradiation was used to simulate the endogenous formation of reductive species, in particular • H atom that is a simple one-electron equivalent reducing agent. Specific damages occur at sensitive amino acid sites, selectively, rather than indiscriminately, leading to the structure modification of the protein. Sulfur-containing residues (Met and Cys) and aromatic residues are appreciably attacked. In particular, extensive changes in the disulfide bridge conformations are induced as well as conformational changes of the protein secondary structure; a gradual conversion of α-helical …to pleated-sheet geometry was evidenced, indicating a higher ability of reducing radicals in denaturing the protein structure compared with that of oxidizing radical species. Show more

Keywords: Bovine pancreatic ribonuclease A, Raman spectroscopy, γ-irradiation, radical damage

DOI: 10.3233/SPE-2008-0350

Citation: Spectroscopy, vol. 22, no. 4, pp. 279-286, 2008

Authors: Ramser, K. | Wenseleers, W. | Dewilde, S. | Van Doorslaer, S. | Moens, L.

Article Type: Research Article

Abstract: Several recent studies on the function of neuroglobin (Ngb), a hemoprotein predominantly expressed in the brain, point toward a neuro-protective role during hypoxic-ischemic injuries. The exact mechanism by which Ngb protects the cell against H2 O2 -induced cell death remains to be elucidated. Hence, new tools need to be developed in order to study the protein in vivo or under physiological conditions. In this summary of our work, we demonstrate how resonance Raman spectroscopy, optical tweezers and microfluidic systems were combined to mimic in vivo conditions in an in vitro milieu. The setup has been tested on several globin-containing cells: …hemoglobin (Hb) within single red blood cells (RBCs), a nerve globin present in the nerve cord of the annelid Aphrodite aculeata (A. aculeata), and wild-type (wt) human neuroglobin (NGB) overexpressed in Escherichia coli (E. coli) bacteria. The feasibility of the setup regarding sensitivity and photo-induced effects and the results regarding the oxygen uptake and release will be discussed and compared for each system. The summary of the results show that the method is promising and the setup will be developed further to monitor the dependence of the neuronal action potential on nerve globins. Show more

Keywords: Neuroglobin, hemoglobin, nerve myoglobin, resonance Raman spectroscopy, optical tweezers, microfluidic system

DOI: 10.3233/SPE-2008-0353

Citation: Spectroscopy, vol. 22, no. 4, pp. 287-295, 2008

Authors: Parker, Stewart F. | Haris, Parvez I.

Article Type: Research Article

Abstract: A combination of infrared, Raman and inelastic neutron scattering (INS) spectroscopies are used to provide complete vibrational spectra of several amino acids and dipeptides. The amino acids studied were glycine, alanine, glutamine, cysteine, methionine and phenylalanine and the dipeptides studied were Gly–Gln and Gly–Ala. The findings of this study have shown how the complementarity of infrared, Raman and INS spectroscopies can be exploited to provide complete vibrational spectra of amino acids and peptides. In particular, the strengths of INS spectroscopy are highlighted: the absence of selection rules, that hydrogenic motions are emphasised, the ready access to the low energy regime …(<400 cm−1 ) and the straightforward calculation of intensities. In the future, it should be possible to apply this approach to the study of larger peptides as well as proteins. Show more

Keywords: Infrared, Raman, inelastic neutron scattering spectroscopy, amino acids, peptides, proteins, density functional theory

DOI: 10.3233/SPE-2008-0354

Citation: Spectroscopy, vol. 22, no. 4, pp. 297-307, 2008

Authors: Amjadi, Mohammad | Manzoori, Jamshid L. | Farzampour, Leila

Article Type: Research Article

Abstract: A new analytical methodology based on the competitive aggregation in a dye–surfactant–drug system is developed for the determination of gemfibrozil. Eriochrome Blue Black R (EBBR) and Didodecyldimethylammonium bromide (DDABr) were the dye and surfactant used, respectively. In the proposed method, the anions of the dye bind to the cationic surfactant molecules to form dye–surfactant aggregates, which are monitored from changes in UV-Vis absorption features of the dye. In the ternary EBBR–DDABr–drug mixtures, the drug competes with the dye to interact with the surfactant, which results in a decrease in dye–surfactant aggregates formation. This, again, causes a change in absorption properties …of the dye. The measurement parameter is the difference between the absorption of the dye in the presence and absence of the drug. In the appropriate experimental conditions the absorbance differences are directly proportional to the drug concentration. The influence of several experimental variables such as pH, concentrations of buffer, EBBR and DDABr on the measurement parameter were studied. Under the optimum conditions, the calibration graph was linear up to 6.0 μg ml−1 with the correlation coefficient of 0.998. The limit of detection and quantification were found to be 0.044 and 0.15 μg ml−1 , respectively. The method was validated and applied to the determination of gemfibrozil in pharmaceutical preparations. Show more

Keywords: Surfactant, mixed aggregate, dye–surfactant aggregate, gemfibrozil

DOI: 10.3233/SPE-2008-0347

Citation: Spectroscopy, vol. 22, no. 4, pp. 309-317, 2008

Authors: Weidemüller, Christian | Hauser, Karin

Article Type: Research Article

Abstract: P-type ATPases are membrane proteins acting as ion pumps that drive an active transport of cations across the membrane against a concentration gradient. The required energy for the ion transport is provided by binding and hydrolysis of ATP. A reaction mechanism of ion transport and energy transduction is assumed to be common for all P-type ATPases and generally described by the Post-Albers cycle. Transient currents and charge translocation of P-type ATPases were extensively investigated by electrical measurements that apply voltage jumps to initiate the reaction cycle. In this study, we simulate an applied voltage across the membrane by an electric …field and perform electrostatic calculations in order to verify the experimentally-driven hypothesis that the energy transduction mechanism is regulated by specific structural elements. Side chain conformational and ionization changes induced by the electric field are evaluated for each transmembrane helix and the selectivity in response is qualitatively analyzed for the Ca2+ -ATPase as well as for structural models of the Na+ /K+ -ATPase. Helix M5 responds with more conformer changes as compared to the other transmembrane helices what is even more emphasized when the stalk region is included. Thus our simulations support experimental results and indicate a crucial role for the highly conserved transmembrane helix M5 in the energy transduction mechanism of P-type ATPases. Show more

Keywords: P-type ATPase, energy transduction, transport, electric field, electrostatics

DOI: 10.3233/SPE-2008-0343

Citation: Spectroscopy, vol. 22, no. 4, pp. 319-325, 2008