Characterisation of Living Matter Using Advanced Spectroscopy Techniques

In the context of an ageing society neurodegenerative disease have become more and more frequent among humans. Opposing the benefits of a longer life, these diseases have thus triggered research on neurons and how they interact with each other. In vitro as well as in vivo experiments are comparably involved. Due to the complex mechanisms occurring at the neuronal level there is a need for developing suitable techniques. Such techniques are required to provide high resolution in several respects in order to track cellular properties: Fourier transform infrared spectroscopy (FTIR) along with high-brilliance synchrotron radiation matches the requirements of high spectral and spatial resolution for chemical distinction perfectly. We have investigated a rat model of Huntington's disease (HD) with synchrotron-assisted Fourier transform infrared microspectroscopy (SIRMS). Several neurodegenerative diseases, ranging from Alzheimer's to prion disease, have now been identified to be caused by proteins which auto-assemble into high molecular weight amyloid aggregates or fibrils. HD is related to abnormal polyglutamine (polyQ) repeats. Its symptoms are progressive deterioration of cognitive and motor functions, along with extensive loss of neurons. In our model, one brain hemisphere was infected with HD while keeping the second one as a control. The two types of matter in the striatum are affected in different manners: in neuron-rich gray matter exposed to the disease, a higher content of aggregated protein is detected, but no signs of cell death. In contrast, myelin-rich white matter did not show any aggregates, but surprisingly showed a significant increase in phosphorylation. We interpret this result as the activation of the cellular response to stress which leads in the end to cell death. The drastic changes in the white matter were detected in the case of multiple sclerosis. We have studied its animal model, the experimental autoimmune encephalomyelitis (EAE). EAE is characterized by heavy loss of the insulating lipid-layer, the myelin around axons. This has been clearly confirmed by SIRMS and FTIR. Analysis of spatially resolved maps by both unsupervised principal component analysis and chemical signatures showed additional features of EAE. Both studies indicate that SIRMS is a powerful tool of detecting complex chemical processes in biological tissue in a relatively easy way. In addition to nanometer spatial resolution, the photonic force microscope (PFM) provides microsecond temporal resolution. A PFM tracks the Brownian motion of micron-sized beads embedded in solutions. Thanks to the high resolutions, we can calculate velocity auto-correlation functions (vacf) of the beads that are extremely sensitive on the short-time scale. We found that they depend in solutions containing polymers on the mesh size. The narrower the polymer mesh size, the earlier perturbations occur in the bead's motion. Polymer solutions possess a very interesting feature which is their viscoelasticity. Based on the mean square displacements of the beads, the complex shear modulus is obtained describing in a frequency-dependent manner viscosity and elasticity. In this way we could distinguish different polymers and also a pathologically relevant fibril: α-synuclein involved in Parkinson's disease.

Influence of the protein environment on spectroscopy and ultrafast dynamics of retinal in bacteriorhodopsin

Selma Schenkl

The environment is important for the exact course of a chemical reaction. As an example of the strong influence of the environment on the reaction, this work studies the isomerization reaction of the chromophore retinal in the binding pocket of the protein bacteriorhodopsin. Three selected distance scales with reference to the chromophore are addressed: The distant protein surface at a length scale of 3-5 nm, the fuzzy environment of the binding pocket (1-2 nm) and the immediate neighborhood represented by the amino acid tryptophan Trp86 (5 Å). First, static absorption and fluorescence spectroscopies are applied to three dimensionally crystallized bacteriorhodopsin. An especially adapted set-up was developed for each task. The analysis of data is carried out in comparison with the measured spectra of the native protein membrane in solution. While the absorption spectrum shows a broadening on the high-energy side, the fluorescence spectrum deviates in the general shape of that measured in solution. The synoptic analysis of all spectra reveals the existence of three spectroscopically distinct species in the crystal: BR490 (absorption maximum at 490 nm) with a relative contribution of 28%, BR570 (native spectrum of solution, 68%), and BR610 ("blue membrane", 4%). BR490 appears particularly in the additional fluorescence band at 550 nm and is assigned to the dehydrated species of bacteriorhodopsin. The presence of that particular species indicates the suppression of rehydration in a partially dehydrated crystal and shows that the three dimensional arrangement hinders water diffusion. A simple model, close domains are assumed to form, eventually suppressing free water diffusion in a crystal. The lack of water on the protein surface modifies the protonation states of the amino acids in the binding pocket and therefore the optical properties of the retinal. The chromophore actually reacts on the alteration of its distant environment upon incorporation in the protein crystal. The second distance scale - the fuzzy environment - is addressed by a time-integrated fluorescence spectroscopy on native bacteriorhodopsin as function of the excitation wavelength (430 nm to 650 nm). The optimized experimental set-up guaranteed that the fluorescence of only the excited state I460 contributed. The measured spectra show maxima at 740 nm, and exhibit a Stokes shift of 5000 cm-1, independent of the excitation wavelength. The similarity of the fluorescence excitation spectrum with the spectrum of absorbed photons excludes energy-dependent loss channels. However, the spectra show a growing asymmetric broadening on the high-energy side with decreasing excitation wavelength. The additional fluorescence originates from vibrational hot states, also supported by the decomposition by Gaussian functions. A comparison with the fluorescence spectrum of blue membranes shows that the low-energy part also arises from vibrational hot states. Consequently, the first fast intramolecular vibrational energy relaxation does not entirely distribute the access-energy to other modes. The remaining energy is found in a quasi-static occupation of vibrational modes. The subsequent second vibrational relaxation — probably not only involving the retinal but also the protein — takes place on a time scale similar to that of the isomerization. The fast isomerization reaction prevents dissipation of energy relaxation of the excited state of the retinal into the protein environment. On a third distance scale, femtosecond spectroscopy in the UV offers a direct view into the electrostatic interaction of the chromophore with its immediate neighborhood. For the first time, the retinal isomerization was studied within the spectral window of the tryptophan absorption from 265 nm to 310 nm. For this, a set-up adapted to UV wavelengths was realized, basing on non-linear optics. The developed single-shot detection system yields a sensitivity of 10-5 in optical density. In the transient absorption measurements, a time resolution of 90 fs was achieved. At short probe wavelengths (265 nm - 294 nm), the measured transients show first a fast reduction of the absorption which subsequently recovers on three time scales (380 fs, 3 ps, > 16 ps). With increasing probe wavelength an induced transient absorption becomes dominant (282 nm - 294 nm). It has a rise time of 280 fs, and decays with a time constant of 4.3 ps. With further increasing probe wavelength the absorption signal decays faster. In response to the complex signature of the transients, an iterative and model-independent transient-decomposition is introduced. A set of three basis elements were identified: one bleach, and two absorption transients. The two induced absorption transients are assigned to retinal. The dynamics of the excited state (I460) in the first retinal-transient is described by two time constants (280 fs and 1 ps). The fast time constant leads to the photoproduct, while the slower one leads back to the ground state, presumably by internal conversion. The 250 fs delayed onset of the second transient (photo-intermediate J) is connected to a wave packet motion in the excited state. This delayed onset of the transient was observed for the first time. It demands a rise time of the J photo-intermediate of only 280 fs. This contrasts common estimates of 500 fs given in literature. For the first time, the temporal behavior of the permanent dipole moment of retinal is observed experimentally. Its evolution is imprinted in the dominant bleach feature (265 nm - 294 nm), originating from tryptophan Trp86. In the excited state, the permanent dipole moment increases strongly within the first 250 fs, and decays exponentially afterwards, following the formation of the photoproduct. However, the related time constant of 380 fs is slightly greater than that of 280 fs of the photoproduct J. This discrepancy may indicate a remaining long-lived polarization of the binding pocket. In brief, by using the intrinsic tryptophan as a probe, the local evolution of the electric field during the isomerization reaction was investigated. Indeed the isomerization reaction is reflected in the immediate environment. The variety of optical spectroscopy provides methods especially adapted for studying the influences of the environment on a chemical reaction. The results achieved here highlight the importance of these influences.

EPFL2004

Self-Assembly of ß-Amyloid Peptides in Cells and on Solid Supports

Enrico Condemi

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease share common mechanisms characterized by protein misfolding and aggregation, including formation of plaques or inclusion bodies. The rapid growing number of patients has motivated intensive research to understand biological phenomena involved in the pathology and hence developing therapeutic strategies. Here, we will focus on β-amyloid peptide (Aβ) involved in Alzheimer's disease. In the first part of this thesis, tissue transglutaminase (tTGase) has been investigated due to its implication in a number of cellular processes and disease states, where the enzymatic actions of tTGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of tTGase in cell survival or cell death mechanisms still remain elusive. tTGase mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that, tTGase induces intramolecular cross-linking of Aβ peptide, resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that, a large population of Aβ monomers contained an increase in mass of 0.984 Da at a glutamine residue, indicating that glutamine Q15 serves as an indispensable substrate in TGase mediated deamidation to glutamate E15. We provide strong analytical evidence on tTGase mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that, tTGase induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation. In the second part of this work, we explore the idea of using synthetic peptides that can switch conformation in a controlled way as a sensor for β-breakers of Aβ. The concept of switch-peptides allows the controlled onset of polypeptides folding and misfolding at physiological pH by in situ O,N-acyl migration. The synthesized sequence consisted in the core sequence responsible for Aβ aggregation, flanked by two β-sheet-inducing template through ester bonds. The triggering of the acyl migration allows the native sequence to be recovered from the ester bonds and structural change from flexible unordered conformation to β-sheets and subsequently fibrils formation were observed in solution. In a more biological environment made of lipid vesicles, the conformation of the peptide has been shown to act comparably to wild type Aβ peptide by circular dichroism (CD), namely increased negative charges over the surface led to increased helical propensity followed by a transition to β-sheets and accelerated fibrils formation. Thereafter, the conformation of the cysteine analogue of the switch-peptide covalently linked to gold surface was characterized by Fourier-transform infrared spectroscopy (FTIR). In monolayer of switch-peptide tethered to a planar gold surface, the peptide already adopts most likely β-turn conformation and no changes in the infrared spectra were detected after the triggering of the switch. On gold nanoparticles, the switch-peptide folds spontaneously into β-sheet conformation. These unwanted foldings limit the efficiency of the sensor and has driven us to an alternative configuration, where gold nanoparticles were coated with a 95:5 mixture of two peptide sequences able to stabilize the nanoparticles and to act as recognition sequence, respectively. In this configuration, the switch-peptide is added in the gold nanoparticles solution and the aggregation triggered. Here, we want to take advantage of a particular feature of gold nanoparticles that consists in a change of solution color from red to blue as the nanoparticles come closer to each other during aggregation. Preliminary results have shown that a discrimination between aggregated and soluble samples is possible. However, further improvements are necessary before a potential biosensor could be expected.

EPFL2011

Hydroxylamine decomposition in the presence of iron studied by calorimetry, online mid-IR and UV-vis spectroscopy

Julien Léo Billeter, Konrad Hungerbühler

Recent explosions at two chemical plants, one in the United States and one in Japan, involving hydroxylamine manufacture underline the lack of mechanistic understanding about this industrially relevant compound. The basic chemical properties of hydroxylamine have been known for decades but its tendency to decompose in the presence of transition metals, in particular Fe(II)/Fe(III), still remains a mystery. Recent studies [1, 2] have not been successful in identifying without controversy the mechanism, products and intermediates of decomposition. In this context we are undertaking a research project to investigate the decomposition of hydroxylamine in aqueous solution in the presence of 3d transition metals. The final aim is to obtain a satisfactory description of the reaction mechanism. A concentration, pH and temperature dependence mechanistic study will be completed using a new pressure-proof small volume reactor [3] that combines calorimetry with online mid-infrared and UV-visible spectroscopy. Chemical models will be fitted to the measured data to determine the reaction mechanism and associated kinetic and thermodynamic parameters [4]. Since there is significant gas production during the decomposition process, the pressure increase inside the reactor will be used as a forth signal. Validation of the online measurements and determination of the chemical species present in the liquid and gas phases will be done by offline GC/MS analysis. Preliminary results for the decomposition of hydroxylamine in the presence of Fe(II)/Fe(III) will be presented including measurements from all the online instrumentation. [1] Cisneros, L. O.; Wu, X. et al. Process Safety and Environmental Protection 2003 81(B2), 121. [2] Wei, C. Y.; Saraf, S. R. et al. Thermochimica Acta 2004, 421(1-2), 1. [3] Visentin, F.; Gianoli, S. I. et al. Organic Process Research & Development 2004, 8(5), 725. [4] Puxty, G.; Maeder, M. et al. Journal of Chemometrics 2005, in print.

2006