Spectroscopy Of Biological Molecules Research Articles

European Conference on the Spectroscopy of Biological Molecules – Dublin 2019

European Conference on the Spectroscopy of Biological Molecules – Dublin 2019

15th European Conference on the Spectroscopy of Biological Molecules (ECSBM) – where spectroscopy and biology met

15th European Conference on the Spectroscopy of Biological Molecules (ECSBM) – where spectroscopy and biology met

Flexibility and dynamics of NhaA Na +/H +-antiporter of Escherichia coli studied by Fourier transform infrared spectroscopy

NhaA (41,355 Da) is a Na +/H + antiporter of Escherichia coli which plays a central role in regulation of intracellular pH, cellular Na + content, and cell volume [E. Padan, S. Schuldiner, J. Exp. Biol. 196 (1994) 443]. Its activity is strongly regulated by pH and increases over 3 orders of magnitude between pH 7 and 8 [A. Rothman, Y. Gerchman, E. Padan, S. Schuldiner, Biochemistry 36 (1997) 14572]. Protein dynamics and flexibility in the activated and inactivated state, respectively, was analysed by probing accessibility in 1H/ 2H exchange experiments for the wild type and the mutant G338S which is constitutively active independent of pH [A. Rimon, Y. Gerchman, Z. Kariv, E. Padan, J. Biol. Chem. 273 (1998) 26470]. This was studied by ATR–FTIR difference spectroscopy using a home built microvolume (<5 μl) perfusion chamber [E. Agić, O. Klein, W. Mäntele, Proceedings of the 10th European Conference on the Spectroscopy of Biological Molecules, vol. 93, 2003, ISBN 9634826148; S. Gourion-Arsiquaud, S. Chevance, P. Bouyer, L. Garnier, J.-L. Montillet, A. Bondon, C. Berthomieu, Biochemistry 44 (2005) 8652]. The solution or suspension of the target protein is contained in a chamber with sample volumes of below 5 μl. It is in contact with the ATR crystal and separated from the flowing effector molecules by a dialysis membrane. The flow-ATR unit is characterised by high stability, fast response, and high sensitivity for the IR spectroscopic detection of binding-induced conformational changes and reactions. On the basis of 1H– 2H exchange of NhaA followed in the amide I and amide II region of the IR spectrum, it is concluded that the accessible fraction of the polypeptide chain of NhaA increases by more than 10% in the active state. For the mutant, no changes in accessibility were observed for different pH values. The increase of Na + concentration increases the extent of exchange. The stability of the wild type protein in the active and inactive form was analysed by measuring the temperature profiles of the IR spectra. A decrease of the structural stability of the protein with activation was observed. Together with the results from 1H/ 2H exchange, the inactive state represents a more compact form whereas activation induces a more open conformation of the protein.

Preface

This special issue contains articles selected from those presented as lectures and posters at the 11th European Conference on the Spectroscopy of Biological Molecules (ECSBM 2005) held in Aschaffenburg, Germany, during September 3–8, 2005. This was the latest in the well-established ECSBM series of international conferences that are organized in alternate years in different European countries to provide a forum for the presentation and discussion of recent progress in the field of biomolecular spectroscopy. Further details on the 2005 meeting remain available on the conference web site at http://www.biophys.uni-frankfurt.de/ecsbmindex.html; the next conference in this series will take place in or near Paris, France, in September 2007. The issue contains 30 papers, all of which have been subject to the thorough review process that is given to any article to be published in Biopolymers. The subject matter covers a broad range of topics in the life sciences where the application of spectroscopic methods has yielded new insights into structure and/or function of the biomolecules studied. The articles are arranged in subsections concerned with biomedical systems and microorganisms (8 papers), structural studies (4), functional studies (6), biomolecules at surfaces (8), and theory/model compounds (4). The range of spectroscopic and other methods employed is similarly broad and encompasses both electronic and vibrational techniques, complemented by others such as atomic force microscopy (AFM), statistical analyses, computer modeling and ab initio molecular orbital (MO) calculations. The full set of articles represents well the state-of-the-art in this fast-moving field, which exploits the ever-increasing sophistication of spectroscopic methods to reveal new insights into both structure and dynamics of biomolecular systems. All of the articles were invited on the basis of a preconference selection from those offered to the organizers and all were rigorously reviewed by at least two referees. For the purpose of this publication, all of the articles were strictly page limited. One of us wishes to use this opportunity to acknowledge the hard work, initiative, and excellent organizational skills of the conference joint-chairmen, Professors Werner Maentele and Friedrich Siebert, and their hard-working local team drawn from their home universities of Frankfurt and Freiburg, respectively. The success of the conference was a tribute to all of them. We also are indeed grateful to all authors and referees for their cooperation and support, which has been essential in enabling us to prepare this issue for publication in a timely manner.

Preface: Biospectroscopy special issue from the 10th European Conference on the Spectroscopy of Biological Molecules (ECSBM 2003)

Preface: Biospectroscopy special issue from the 10^th European Conference on the Spectroscopy of Biological Molecules (ECSBM 2003)

Infrared microspectroscopy mapping studies of packaging materials: experiment design and data profiling considerations

Over the past decade, infrared microspectroscopy mapping has been applied to a wide variety of sample types [H.J. Humecki (Ed.), Practical Guide to Infrared Microspectroscopy, Marcel Dekker, New York, 1995.], including polymers [J.A. Reffner, in: P.E. Russell (Ed.), Microbeam Analysis, San Francisco Press, San Francisco, 1989, p. 167.], wheat kernels [D.L. Wetzel, J.A. Reffner, Cereal Foods World 38 (1) (1993) 9.], painted manuscripts [M.R. Derrick, Mater. Res. Soc. Symp. Proc. 352 (1995) 97.], and carcinoma tissue sections [P. Lasch, D. Naumann, in: P. Carmona (Ed.), Spectroscopy of Biological Molecules: Modern Trends, Kluwer Academic Publishers, Netherlands, 1997, p. 441.]. However, little has been mentioned regarding proper mapping conditions and data treatment. Proper experiment design (sample preparation, map definition) and data treatments (viewing formats, profile creation) are essential for success, especially if the sample contains features that are smaller than the diffraction limit (10 μm at 1000 cm −1). The data obtained from line maps is especially susceptible to misinterpretation if not handled properly. For example, diffraction is a well-known property that every infrared microspectroscopist is aware of, yet its effect on mapping experiments, especially when it comes to creating profiles is not documented and is often neglected. This paper will discuss how mapping experiments and profiles can be successfully used to extract useful and valid data from line maps. Examples will be discussed of two challenging line map samples: finding a thin adhesive layer, and the reverse-engineering of a complex multi-layer laminate.

Intracellular fate of oligodeoxynucleotides: A microspectro FRET study

Fluorescence resonance energy transfer (FRET) under confocal microscopy allows direct intracellular measurements of both localisation and interactions of tagged molecules to be performed. In the antigene and antisense strategies, short oligonucleotide sequences are used to inhibit RNA translation in cell. Numerous techniques (Crook, R.M. “In Vitro Cellular Uptake, Distribution, and Metabolism of Oligonucleotides” Handbook of Experimental Pharmacology, Vol. 131 Antisense Research and Application : 103-140 (1998) were used to follow oligonucleotide penetration and distribution in cultured cells, but no in situ study of the oligonucleotide stability was performed. Therefore, microspectroFRET was chosen as a non destructive method to study in differents cell lines the fate of short oligonucleotides, fluorescently labeled at both ends. When an oligonucleotide is intact, FRET occurs, so, in this study, intramolecular FRET permits us to know the ratio of intact oligonucleotides during time, i. e. the ratio of potentially active oligonucleotides ; single fluorescent labeling is not sufficient : the observed fluorescence may be due to fluorophores alone or still grafted on the oligonucleotide. The oligonucleotides, labeled with fluorescein at the 3’ end and rhodamine at the 5’ end, were added to the cells and subcellular microvolumes (-10-18 cubic meter) were analysed with the confocal microspectrofluorometer (Vigny, P. Amirand, C. Ballini, J.P. Chinsky, L. Duquesne, M. Laigle, A. Manfait, A. Sureau, F. Turpin, P.Y. “Microspectrofluorometry of Single Living Cells” Spectroscopy of Biological Molecules - State of the Art : 345-348 (1989) developed in the laboratory. Different wavelengths of an Argon LASER were used for excitation of the samples. The spectra were recorded on an optical multichannels analyser after separation of the different emission wavelengths by a monochromator. After FFT smoothing and background subtraction, comparisons of the spectra were achieved and analysed in term of : Stability of the oligonucleotides, which is directly related to the transfer efficiency, as a function of time Quantitative localisation of the oligonucleotides, as fluorescence intensity is proportional to concentration Surrounding of the oligonucleotides, as the quatum yield of fluorescein is pH dependent. As an example of granulocytic cell model, we used K562 cells. But these cells have a large nucleus, preventing to distinguish subcellular compartments. So we also used Hela cells as an example of epithelial-like cells with a real differentiation between cytoplasm and nucleus. 3e Colloque SFp, Paris-Sud ‘99 icole Polytechnique, Palaiseau, 28 juin-2 juillet 1999

Spectroscopy of Biological Molecules. (6th European Conference on the Spectroscopy of Biological Molecules, 3-8 September 1995, Villeneuve d'Àscq, France)

Spectroscopy of Biological Molecules. (6th European Conference on the Spectroscopy of Biological Molecules, 3-8 September 1995, Villeneuve d'Àscq, France)

September 1–6, 1991 4th European conference on the spectroscopy of biological molecules

September 1–6, 1991 4th European conference on the spectroscopy of biological molecules

4th European conferencw on the spectroscopy of biological molecules, York, U.K., September 1-6, 1991

4th European conferencw on the spectroscopy of biological molecules, York, U.K., September 1-6, 1991

4th biennial European conference on the spectroscopy of biological molecules

4th biennial European conference on the spectroscopy of biological molecules

Fast time-resolved i.r. spectroscopy of biological molecules in aqueous solution: The reaction kinetics of myoglobin with carbon monoxide

The recombination of myoglobin (Mb) and CO, generated by the u.v. flash photolysis of carboxymyoglobin (MbCO) is monitored by time-resolved i.r. spectroscopy (TRIR) on a microsecond timescale. The TRIR measurements are made on the ν(CO) i.r. band of MbCO centered at 1945 cm −1. Experiments have been performed over a wide range of concentrations of both Mb and CO and the rate constants derived are in excellent agreement with those obtained by conventional flash photolysis. The results represent a significant improvement in sensitivity over previous i.r. experiments of this type.

FT-Raman spectroscopy of biological molecules

Raman spectroscopy of biological molecules is often very difficult if not impossible due to a large fluorescence background from absorbing species, either from the molecule itself or an impurity. Photobleaching is occasionally successful in photochemically removing fluorescent impurities, but the majority of samples are not responsive to such treatment. Resonance enhancement of an absorbing species allows acquisition of Raman spectra in spite of competing fluorescence. However, the resonance Raman spectrum is characteristic of the chromophore only and little structural information is obtained from the spectrum about other parts of the molecule which are not resonantly enhanced. The newly developed technique of FT-Raman spectroscopy proves to be a solution to both of these problems for biological materials. Excitation with infrared wavelengths prevents electronic absorptions which give rise to fluorescence. In addition, the obtained spectra are completely nonresonant, allowing detection of vibrational modes of all parts of the molecule including the chromophore. We will present nonresonant, fluorescence free spectra of a range of biologically significant molecules including phospholipids and porphyrins.

A Raman spectroscopic study of the interaction between nucleotides and the DNA binding protein gp32 of bacteriophage T4.

AbstractRaman spectra of the bacteriophage T4 denaturing protein gp32, its complex with the polynucleotides poly(rA), poly(dA), poly(dT), poly(rU), and poly(rC), and with the oligonucleotides (dA)8 and (dA)2, were recorded and interpreted. According to an analysis of the gp32 spectra with the reference intensity profiles of Alix and co‐workers [M. Berjot, L. Marx, and A. J. P. Alix (1985) J. Ramanspectrosc., submitted; A. J. P. Alix, M. Berjot, and J. Marx (1985) in Spectroscopy of Biological Molecules, A. J. P. Alix, L. Bernard, and M. Manfait, Eds., pp. 149–154], 1 gp32 contains ≈ 45% helix, ≈ 40% β‐sheet, and 15% undefined structure. Aggregation of gp32 at concentrations higher than 40 mg/mL leads to a coordination of the phenolic OH groups of 4–6 tyrosines and of all the sulfhydryl (SH) groups present in the protein with the COO− groups of protein. The latter coordination persists even at concentrations as low as 1 mg/mL. In polynucleotide–protein complexes the nucleotide shields the 4–6 tyrosine residues from coordination by the COO− groups even at high protein concentration. The presence of the nucleotide causes no shielding of the SH groups. With Raman difference spectroscopy it is shown that binding of the protein to a single‐stranded nucleotide involves both tyrosine and trytophan residues. A change in the secondary structure of the protein upon binding is observed. In the complex, gp32 contains more β‐sheet structure than when uncomplexed. A comparison of the spectra of complexed poly(rA) and poly(dA) with the spectra of their solution conformations at 15°C reveals that in both polynucleotides the phosphodiester vibration changes upon complex formation in the same way as upon a transition from a regular to a more disordered conformation. Distortion of the phosphate–sugar–base conformation occurs upon complex formation, so that the spectra of poly(rA) and poly(dA) are more alike in the complex than they are in the free polynucleotides. The decrease in intensity of the Raman bands at 1304 cm−1 in poly(rA), at 1230 cm−1 in poly(rU), and at 1240 and 1378 cm−1 of poly(dT) may be indicative of increased stacking interactions in the complex. No influence of the nucleotide chain length upon the Raman spectrum of gp322 in the complex was detected. Both the nucleotide lines and the protein lines in the spectrum of a complex are identical in poly(dA) and (dA)8.

BOOK REVIEWS

Book reviewed in this article: Spectroscopy of Biological Molecules, Edited by C. Sandorfy and T. Theophanides. Specificity in Biological Interactions, Edited by C. Chagas and B. Pullman. Solar‐UV Actions on Living Cells, by John Jagger