Ebook: Spectroscopy of Biological Molecules

The 14th European Conference on the Spectroscopy of Biological Molecules was held in the campus of the University of Coimbra, Portugal, from 29th August to 3rd September 2011. The local organisers of the conference were Maria Paula Marques and Luis A.E. Batista de Carvalho. The University of Coimbra, founded in 1270, is one of the oldest in the world. It provided an excellent venue for spectroscopists from Europe and other parts of the world to meet, present their latest research work and engage in a fruitful exchange of ideas. Some of the topics discussed in the conference are presented in this book.

This special volume, containing the Proceedings of the 14th European Conference on Spectroscopy of Biological Molecules (28 August–3 September 2011, Coimbra, Portugal), is dedicated to Professor Juana Bellanato, who is an inspiration to us all. We all had the pleasure of meeting her in Coimbra. One of us (PIH) previously communicated with Prof. Bellanato to obtain photograph of her, next to an early commercial infrared spectrometer, for inclusion in a book (see [1]). Prof. Bellanato is one of the early pioneers in the use of vibrational spectroscopy for the study of biological molecules and her work has been highlighted by Barth and Haris [1]. Maria Paula Marques (MPM), one of the organisers of the conference, had a conversation with Prof. Bellanato (JB) in Coimbra and a summary of this is presented below.

Prof. Juana Bellanato obtained her BSc in Chemistry, in 1950, and her PhD in Chemistry in 1954 (both from the Complutense University of Madrid, Spain).

She started her academic career in the Spanish National Research Council (CSIC, Madrid) as a research assistant at the “Patronato Alfonso X el Sabio”, in 1955, and as a scientific collaborator of the “Patronato Juan de la Cierva”, in 1956.

She obtained postdoctoral fellowships for the Institute of Physical-Chemistry of the University of Freiburg (Germany), between 1956 and 1957, and for the Physical-Chemistry Laboratory of the University of Oxford (UK), from 1959 to 1960.

In 1967, she became a research scientist at the “Patronato Juan de la Cierva”, and from 1971 to 1990 she was a research professor at the Institute of Optics of CSIC (Madrid). Between 1990 and 1994 she was Doctor “ad honorem” at the Institute of Optics. Since 1994, she is a Doctor “ad honorem” at the Institute of Structure of Matter of CSIC.

She is the author of more than 200 scientific papers, 1 book, and several chapters in books. She has also collaborated with numerous international groups, with short stays in research centers from Montreal, Florence and Bologna Universities.

She was appointed member of numerous scientific boards:

1975–1979 Head of the Molecular Spectra Section and of the Infrared Spectroscopy Laboratory of the Institute of Optics of CSIC.

1979–1990 Head of the Molecular Spectroscopy Department of the Institute of Optics of CSIC.

1985–1988 President of the Spanish Spectroscopy Committee and Vice-president of the Spanish Spectroscopy Group.

1990–1994 President of the Spanish Spectroscopy Group.

Since 1987 Member of the International Organizing Committee of the European Congress on Molecular Spectroscopy (EUCMOS).

She was awarded several prizes:

1968 Perkin Elmer Prize to the best work on Absorption Spectroscopy (with A. Hidalgo).

1996 Silver Medal of the Spanish Spectroscopy Committee.

2002 Honor Fellow and Silver Medal of the Spanish Optical Society.

2003 Medal of the Spanish Royal Society of Chemistry.

2006 Prize “Jesús Morcillo Rubio” of the Iberic Spectroscopy Conference (CIE) (sponsored by Bruker).

2006 Institution Award of CSIC.

2007 Gold and Brilliant Insignia of the Chemists' Association of Madrid.

Reference

[1] A. Barth and P. Haris, Infrared spectroscopy – Past and present, in: Biological and Biomedical Infrared Spectroscopy, A. Barth and P.I. Haris, eds, IOS Press, Amsterdam, 2009, pp. 1–52.

Maria Paula Marques, Luis A.E. Batista de Carvalho and Parvez I. Haris

This review summarizes the progress achieved over the past fifteen years in applying vibrational (Raman and IR) spectroscopy to problems of medical diagnostics and cellular biology. During this time, a number of research groups has verified the enormous information content of vibrational spectra; in fact, genomic, proteomic and metabolomic information can be deduced by decoding the observed vibrational spectra. This decoding process is aided enormously by the availability of high power computer workstations, and advanced algorithms for data analysis. Furthermore, commercial instrumentation for the fast collection of both Raman and infrared microspectral data has rendered practical the collection of images based solely on spectral data. The progress in the field has been manifested by a steady increase in the number and quality of publications submitted by established and new research groups in vibrational biological and biomedical arenas.

The increasing bacteria resistance to conventional antibiotics has led to the need for alternative therapies. Being part of the human innate defence system and with a broad spectrum of activity against bacteria, viruses, protozoa and cancer cells, antimicrobial peptides (AMPs) are a very promising alternative. The mechanism of action of AMPs seems to broadly correlate with their ability to target the bacterial cell membrane. To understand and improve their effect, it is of major importance to unravel their mechanism of action and in particular, to understand the peptide–membrane binding. Several biophysical techniques such as fluorescence spectroscopy, circular dichroism, zeta potential determination and atomic force microscopy can be used to achieve this goal. Characteristics of AMPs interactions and the use of these biophysical techniques will be discussed.

Uptake of the β-blocker drug propranolol by living glial C6 cells has been observed using fluorescence lifetime imaging with two-photon excitation at 630 nm. Both uptake and release of propranolol occur within minutes and are temperature dependent, being about 5 times faster at 37°C than at 20°C. The intracellular fluorescence lifetime of propranolol is generally shorter than the value of 9.8 ns determined in dilute neutral aqueous solution and the difference is ascribed to concentration quenching. Within the cells propranolol is accumulated within intracellular acidic vesicles and the cytoplasm, but is excluded from the cell nucleus. On incubation of cells in medium containing 100 μM propranolol, the drug is accumulated to reach intracellular concentrations up to 10 mM in a process that is believed to be driven by protonation within acidic cellular compartments.

Fungi are considered as serious pathogens for many plants, potentially causing severe economic damage. Early detection and identification of these pathogens is crucial for their timely control. The methods available for identification of fungi are time consuming and not always very specific. In this study, the potential of FTIR-ATR spectroscopy was examined together with advanced mathematical principle component analysis (PCA) and statistical linear discriminant analysis (LDA) to differentiate among 10 isolates of Fusarium oxysporum. The results are encouraging and indicate that FTIR-ATR can successfully detect different isolates of Fusarium oxysporum. Based on PCA and LDA calculations in the region 850–1775 cm⁻¹ with 16 PCs, the different strains from the same fungal genus could be classified with 75.3 and 69.5% success rates using the “leave one out” method and “20–80% algorithm” respectively.

In this work we describe the specific spectral signature of different phospholipids and sphingolipids in the far infrared. Three specific spectral domains have been found: the head group contributions (600 and 480 cm⁻¹); the modes of the torsion motion of the hydrocarbon chains and of the skeleton vibration (460 to 180 cm⁻¹); and the hydrogen bonding continuum (below 300 cm⁻¹). Marker bands for individual phospholipids are distinguished.

Folding dynamics for β-structure loss and disordered structure gain were studied in a model β-hairpin peptide based on Cochran's tryptophan zipper peptide Trpzip2, but with an altered Thr–Gly (TG) turn sequence, i.e. SWTWETGKWTWK, using laser induced temperature-jump (T-jump) kinetics with IR detection. As has been shown previously, the TG turn sequence reduces the thermodynamic β-hairpin stability as compared to the Asn–Gly sequence used in Trpzip2 (TZ2-NG). In this study, we found that the TG-turn slows down the overall relaxation dynamics as compared to TZ2-NG, which were studied at higher temperatures where the time constants show little difference between relaxation of the β-strand and the disordered conformation. These time constants become equivalent at lower temperatures for TZ2-TG than was seen for TZ2-NG. The correlation of thermodynamic stability and rates of relaxation suggests that the change from NG to TG turn results in a slowing of folding, lower k_f, with less change of the unfolding rate, k_u, assuming two state behavior at higher temperatures.

Microorganisms require water for their metabolic activities. Only a fraction of water in foodstuffs, the so-called “free water”, is available for this purpose. The amounts of “free” water previously estimated by two different methods [J. Phys. Chem. A 114 (2010), 11933–11942; J. Phys. Chem. A 155 (2011), 4563–4563; Journal de Recherches Atmosphériques 4 (1969), 65–78] are compared for aqueous solutions of four electrolytes, NaCl, NH₄Cl, Na₂SO₄, (NH₄)₂SO₄: (i) Vapour pressure measurements of the solutions relative to that of pure water (water activities). (ii) Low-wavenumber Raman spectra in the R(ν)-representation. For each electrolyte deviations were found between results from the two methods. All water molecules in the illuminated volume contribute to the Raman data. The vapor pressure measurements refer to water molecules at the water/atmosphere interface where surface tension is important. Differences in surface tension for the four electrolytes qualitatively explain deviations between the amounts of “free water” observed by the two methods.

In the present work, real ability of a confocal Raman microspectroscopy to monitor chemical composition of the vacuoles within living yeast cells was investigated and critically assessed. Simple, economical and practical protocols of the yeast immobilization suitable for less laborious, high-throughput and spatially-resolved Raman measurements were tested for their possible impacts on physiological states and viability of the cells. We have demonstrated that acquiring Raman spectra from statistically sound sets of immobilized cells and employing advanced multivariate methods for spectral analysis, the chemical composition of the yeast vacuoles can be reliably studied. The most easily and accurately quantifiable seems to be the concentration of polyphosphates which can be unambiguously identified due to unmistakable Raman features. Our approach can be useful for routine, label-free and non-invasive monitoring of the chemical composition of the vacuoles of living yeasts exposed to various stress factors, the information important in biomedical research of pathogens.

Halictine-1 (Hal-1) – a linear antibacterial dodecapeptide isolated from the venom of the eusocial bee Halictus sexcinctus – has been subjected to a detailed spectroscopic study including circular dichroism, fluorescence and vibrational spectroscopy. We investigated Hal-1's ability to adopt an amphipathic α-helical structure upon interaction with model lipid based bacterial membranes (phosphatidylcholine/phosphatidylglycerol based large unilamellar vesicles, sodium dodecylsulfate micelles) and helix inducing components (trifluoroethanol). It was found that Hal-1 responds sensitively to composition of the membrane model and to peptide/lipid ratio. Amphipathic nature of the helical Hal-1 seems to favour flat charged surfaces of the model lipid particles over the non-directional interaction with trifluoroethanol. Increasing fraction of polyproline II type conformation was detected at low peptide/lipid ratios.

Methimazole (2-mercapto-1-methylimidazole, MMI) and its pentacyanoferrate(II) complex have been characterized by means of UV/Vis, Raman and NMR spectroscopy. The reaction of MMI with aquapentacyanoferrate(II), [Fe(CN)₅(H₂O)]³⁻, in buffered aqueous solutions yielded the [Fe(CN)₅(MMI)]³⁻ complex. It was found that only thione tautomer of MMI coordinates to the iron(II). The exceptionally stabile and inert complex was produced.

This paper describes a metabonomics study of 2nd trimester biofluids (amniotic fluid, maternal urine and blood plasma), in an attempt to correlate biofluid metabolic changes with suspected/diagnosed fetal malformations (FM) and chromosomal disorders, as well as with later occurring gestational diabetes mellitus (GDM), preterm delivery (PTD) and premature rupture of membranes (PROM). The global biochemical picture given by the threesome of biofluids should enable the definition of potential disease signatures and unveil potential metabolite markers for clinical use in predictive prenatal diagnostics.

Results show that relatively strong metabolic disturbances accompany FM, reflected in all three biofluids and thus suggesting the involvement of both fetal and maternal metabolisms. Regarding GDM, amniotic fluid and maternal urine seem potential good media to detect early metabolic changes and PTD subjects show small metabolite changes in the same biofluids, undergoing work being focused on plasma composition. Chromosomal disorders show an interestingly marked effect on maternal urine whereas no statistically relevant early changes have been observed for PROM subjects. Interestingly, in the case of FM and chromosomal disorders, maternal biofluids show some sensitivity to disorder type e.g. for central nervous system malformations and trisomy 21, respectively. These results show the usefulness of biofluid metabonomics to probe overall metabolic disturbances in relation to prenatal disorders.

Drop-coating deposition Raman (DCDR) spectroscopy was employed to study liposome suspensions. The method is based on a specific drying process on the hydrophobic surface that efficiently accumulates the macromolecular sample in a ring of the edge of the dried drop. We studied liposome suspensions purchased from two sources (Avanti Polar Lipids, Inc. and Sigma-Aldrich, Co.) and prepared under different conditions. Structure of the dried drop substantially depends on the lipid concentration, lipid composition of the sample and used solvent. Optimal lipid concentration is about 0.3 mg/ml in all cases, asolectin and DSPC suspensions form compact dried drops when dissolved in water and phosphate buffer, respectively. Drying process of the sample drop does not influence the initial phase state (gel or liquid-crystalline) of the studied liposomes excepting DSPC from Sigma-Aldrich, Co.

Type I and IV collagens are important constituents of the skin. Type I collagen is found in all dermal layers in high proportion while type IV collagen is localized in the basement membrane of the dermo-epidermal junction (DEJ). These proteins are strongly altered during aging or cancer progression. Although they possess amino acid compositions which are close, they present also important structural differences inducing specific physicochemical properties. Raman spectroscopy is based on a non-destructive interaction of the light with the matter. This technique permits to probe the intrinsic molecular composition of the samples without staining nor particular preparation. The aim of our research is to study the correlation between the molecular conformations of the type I and IV collagens and their Raman features. We showed that signals specific of each protein can be revealed and that they translate structural differences between the two collagens. From this collagens spectral characterization, the analysis of skin sections also permitted to identify spectral markers of dermis, epidermis and epidermis/dermis interface. These preliminary results represent basic data for further studies, particularly to probe skin molecular alterations induced by chronologic aging.

Among the optical characterization techniques, the Fourier Transform Infrared spectroscopy (FTIR) can offer high sensibility and accuracy to detect minimal chemical changes into the biological sample and in principle, can be used to differentiate the normal from neoplastic tissues. The aim of the present study is to evaluate the differences on FTIR spectra of the normal cell due to different fixation protocols for histological processing. Immortalized, non-cancerous mice lung epithelial cell line e10 (American Type Culture Collection, Rockville, MD, USA) was maintained in complete e10 culture medium (CMRL-1066 (Gibco), 10% SUF and L-glutamine 200 mM). The cultures were fixed with following substances: 5% formalin in PBS, methacarn (60% methanol, 30% chloroform and 10% acetyl acid), 70% alcohol and also the unfixed cells, were cultured and maintained in PBS. The FTIR spectra were acquired on a Nicolet 6700 (Thermo Scientific Nicolet™, Waltham, MA, USA) spectrophotometer at 4 cm⁻¹ resolution, 30 scans, in the 4000–500 cm⁻¹ spectral range. Nine infrared spectra were obtained from each fixation protocol. Main region of spectra used in the analysis was 1800–800 cm⁻¹. All spectra were analyzed by vector normalization, to determine any possible difference in bands and peak position. These results indicate that all fixing protocol change some of the specific characteristics of FTIR spectra. The formalin fixation was the protocol that caused less modification on the spectra related to the unfixed one. Therefore the choice of the fixing protocol depends on the specific information wanted from the spectra.

The role of water in the behaviour of biomolecules is well recognized. The coupling of motions between water and biomolecules has been studied in a wide time scale for the self part while collective dynamics is still quite unexplored. Self dynamics provide information about the diffusion processes of water molecules and relaxation processes of the protein structure. Collective density fluctuations might provide important insight on the transmission of information possibly correlated to biological functionality. The idea that hydration water layers surrounding a biological molecule show a self dynamical signature that differs appreciably from that of bulk water, in analogy with glass-former systems, is quite accepted. In the same picture Brillouin Terahertz spectroscopy has been used to directly probe collective dynamics of hydration water molecules around bio-systems, showing a weaker coupling and a more bulk-like behaviour. We will discuss results of collective modes hydration water, arising from neutron Brillouin spectroscopy, in the context of bio-molecules-solvent interaction.

The present work reports a conformational study of solid-state anhydrous guanine, using vibrational spectroscopy techniques – infrared, Raman and inelastic neutron scattering – coupled to quantum mechanical methods at the DFT level, both for the isolated molecule and the condensed state. In both cases, the 7H-keto-amino tautomer was found to be the prevalent form, contrary to aqueous solutions and hydrated polycrystalline guanine, where the 9H-keto-amino tautomer is the most favoured species. This study is a significant contribution for the existing spectroscopic characterisation of this purine base, by unambiguously assigning its vibrational spectra.

A conformational analysis of the Pt(dap)Cl₂ complex (dap = 1,3-diaminopropane) was performed by vibrational spectroscopy (FTIR, Raman and INS), coupled to quantum mechanical methods within the Density Functional Theory (DFT) and Effective Core Potential (ECP) approaches.

A complete spectral assignment of the system was achieved, due to the combined use of all available vibrational spectroscopic techniques. A good agreement was found between experimental and theoretical results, as well as with reported data for analogous complexes (e.g., cisplatin).

Thymoquinone is a natural product, the main constituent of Nigella sativa seeds, which exhibits anti-inflammatory and anti-cancer activities. Among several existing molecules capable of forming an inclusion compound structure, cyclodextrins are applied in the pharmaceutical industry either to increase solubility of hydrophobic molecules or to protect molecules from inactivation or degradation. β-cyclodextrin is currently the most common cyclodextrin in pharmaceutical formulations and probably the best studied in humans. In order to study the properties of inclusion compounds based on cyclodextrins and thymoquinone Fourier Transform Infrared (FTIR), Utraviolet-Visible, Positron Annihilation Lifetime (PAL) spectroscopies and calorimetric studies by Differential Scanning Calorimetry (DSC) were used. The obtained results indicate the formation of a 1:1 inclusion compound between cyclodextrin and thymoquinone. PALS and DSC measurements also provided evidence of the inclusion compound's activity.

In this article, we describe a new method to obtain D₂O/H₂O exchange in photosynthetic reaction centres from Rhodobacter sphaeroides. The method is characterized by: (i) a very high efficiency of the isotopic replacement; (ii) an extremely low amount of D₂O needed; (iii) the short time required for dehydration and D₂O rehydration; (iv) the possibility of controlling concomitantly the hydration state of the sample. The proposed method can be applied to other proteins.

We report the application of dried Ag hydroxylamine-reduced colloidal drops to surface-enhanced (resonance) Raman scattering (SE(R)RS) study of biomolecules using Raman microspectroscopy. 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (TMPyP), amino acid tryptophan and phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) served as testing biomolecules. Ag colloid/biomolecule drop dried on glass support forms a ring in the edge part of the drop in which almost all nanoparticles are clustered. This specific drying process promotes adsorption of the studied biomolecules in highly enhancing sites (‘hot spots’) as well as concentrates them in the ring. We were able to obtain SE(R)RS spectra from the ring that cannot be acquired directly from Ag colloidal solutions (SERRS spectrum of 1×10⁻¹⁰ M TMPyP by 1 s accumulation time, SERS spectrum of 2×10⁻⁷ M DSPC). Despite the spectral irreproducibility and problems with spurious bands in some cases, SERS microspectroscopy of studied biomolecules using dried Ag colloid/adsorbate systems improves SERS applicability and sensitivity in comparison to measurements directly from Ag colloidal solution.

The interaction of the linker histone H1Z from the sperm chromatin of starfish Asterias amurensis with DNA was studied by spectroscopic and thermodynamic approaches. It has been shown that at the physiological conditions the interaction of the H1Z with DNA results in more compact structures compared to complexes of DNA with somatic histone H1. The typical profile of the DNA melting curves reveals two peaks attributed to the bound and unbound DNA. It has been shown that H1Z from starfish sperm stabilizes DNA to a greater extent compared to the somatic H1. It is possible that the presence of the additional α-helical segments within the C-terminal part of the H1Z typical for the linker histones from echinoderm sperm facilitates the protein–protein interactions which in turn stimulate co-operative binding of the histones to DNA, resulting in the formation of the super compact sperm chromatin.

The structural organization of the DNA complexes with non-histone chromosomal protein and linker histone H1 was studied using circular dichroism spectroscopy (CD) and atomic force microscopy (AFM). It has been shown that due to the interaction between HMGB1 and H1 highly ordered DNA–protein complexes emerge in the solution. Their spectral properties are found to be similar to those of DNA/HMGB1-(AB) complexes, reported earlier. AFM images reveal the formation of fibril-like structures in the solution. We suggest, that the electrostatic screening of the HMGB1 C-terminal domain by histone H1 facilitates stronger interaction of the HMGB1/H1 with DNA and the formation of the ordered supramolecular DNA–protein complexes.

Fluorescence micro-imaging and homodyne phase-resolved confocal microspectrofluorimetry were used to monitor the transport of antisense oligonucleotide into cancer MCF7 cells and the subsequent intracellular distribution. Phosphorothioate analog of 15-mer oligoadenylate (dA₁₅) labeled by ATTO 425 was complexed with 5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin (H₂TMPyP4) as an uptake-mediating agent. Fluorescence lifetime data within a broad spectral range have revealed properties of both components inside the cell. H₂TMPyP4 lifetime inside the cell is not influenced in this malignant cell line, while the lifetime of modified oligonucleotide was found to be slightly shortened.

We investigated distribution of phosphorus under manganese toxicity in different tissues of 10 μm thin root cross-section of Douglas fir (DF) (Pseudotsuga menziesii) seedlings by using synchrotron based Fourier transform infrared microscopy (SR-FTIR) as a chemically sensitive imaging method. Manganese is an essential micro-nutrient in all organisms but may become toxic when present in excess. We found previously that DF variety glauca (DFG) and variety menziesii (DFM) differed in phosphorus uptake, subcellular localization, transport and tissue allocation, as an effect of manganese toxicity. To address the role of P in seedling tolerance under Mn toxicity we determined P allocation in different root tissues. In DFG, but not in DFV, the P concentration was kept at a constant level even under Mn toxicity. Earlier X-ray microanalysis showed Mn accumulation in epidermal and cortical cells of both varieties after Mn treatment, suggesting the root endodermis was a barrier for Mn to protect the vascular system and shoot from high Mn, with possible role of P ameliorations. Here we discuss the potential role of P in Mn compartmentalization and toxicity tolerance in two different varieties.