Investigation of Physiological Solutions of Metalloproteins in a High-Repetition Rate Picosecond X-ray Absorption Experiment

Over the last decade, ultrafast time-resolved X-ray absorption spectroscopy (XAS) has evolved to be now a mature and well-established experimental technique, giving extremely detailed information about the local geometrical and electronic structure in the early stages of a chemical reaction or biological process. Electronic structure changes are the fundamental driving forces triggering structural modifications in many chemical and biological reactions. Ultrafast time-resolved XAS is an ideal experimental technique to probe these changes in "real time" during the course of a chemical reaction, a biological function or a physical process. Ideally, the observation of ultrafast processes is made in conditions as close as possible as the natural ones, i.e., biological processes in physiological media (instead of crystals or frozen films). In this sense XAS offers unique capabilities, since it can be applied to any kind of system. In this thesis we successfully implemented a new scheme for measuring time-resolved XAS spectra with picosecond temporal resolution at MHz repetition rate. The increase in the data acquisition repetition rate provided an increase in the signal-to-noise (S/N) of a factor of > 20 compared to previous experiments in the kHz regime. To assess the improvements in this new data acquisition methodology, we used the light-induced spin transition in aqueous solutions of [Fe(bpy)3]2+ to benchmark our experiments. This system has been well-characterized by both ultrafast laser and x-ray time-resolved spectroscopies. The expected gain in data quality was confirmed, which allowed the recording of subtle changes in the pre-edge region of the XAS spectrum, reflecting the different electronic structure of [Fe(bpy)3]2+ upon the spin transition. We also present the investigation of the electronic and geometric structures of a series of metalloproteins (Myoglobin) in physiological solutions by means of X-ray absorption spectroscopy. To our knowledge, this is the first study of the structure of the different forms of Myoglobin in physiological media using XAS. The analysis of the XANES region of the spectrum using full-multiple scattering (FMS) formalism revealed that the ironnitrogen bond length in the porphyrins ring converged to a common value of about 2 Å, in contrast to the wide variation found in the crystallographic structures. Porphyrins are known to be very rigid structures due to the big number of carbon-carbon double bounds, supporting our conclusion. The relative geometry of the ligands with respect to the heme is reported for the whole series of Myoglobins investigated. In addition, time-resolved XAS has been measured for two types of Myoglobin, Carboxy-Myoglobin (MbCO) and (Nitrosyl-Myoglobin) MbNO. It has been shown that, as expected from previous studies, the transient structure of photo-excited MbCO resembles that of the deligated ferrous Myoglobin (deoxyMb). On the other hand, the transient structure of photo-excited MbNO at 50 ps deviates slightly from that of deoxyMb. The analysis of the transient XANES indicates that the NO molecule moves 2.88 Å away from the heme, staying closer to the iron atom than in the case of photo-excited MbCO. The NO geminate recombination time with the heme was also measured in "real time", and it has been found to occur in 216 ± 24 ps. This is the first direct measurement, i.e. sensitive to structural changes, of this geminate recombination time.

Atmospheric heterogeneous reactions of chlorine and bromine containing molecules

Arnaud Aguzzi

This thesis deals with a laboratory study of heterogeneous reactions involving bromine and chlorine containing reservoir species on alkali salt substrates which are relevant to the marine boundary layer, and on ice substrates which are relevant to the lower stratosphere. The aim is to obtain reliable data on uptake coefficients γ and reaction mechanisms for the sake of extrapolation of results obtained in the laboratory to atmospheric conditions. The experiments have been performed in a Teflon coated Knudsen flow reactor equipped with a quadrupole mass spectrometer. The reactions of BrONO2 with solid alkali halides have been studied at ambient temperature. The initial uptake coefficients γ0 of BrONO2 on NaCl and KBr substrates are γ0 = 0.31 ± 0.10 and γ0 = 0.32 ± 0.09, respectively. For NaCl substrates BrCl, Br2 and HCl and for KBr both Br2 and HBr are observed as products. HCl and HBr result from the interaction of NaCl and KBr, respectively, with HNO3 generated in the hydrolysis of BrONO2 with H2O condensed on the salt sample. Using NaCl we observed Br2 which is formed from heterogeneous BrONO2 decomposition occurring on solid KBr samples. Halogen exchange reactions are competing with hydrolysis and decomposition which also take place on non-reactive salts (NaNO3, Na2SO4). A high rate of initial uptake (0.2 ≤ γ0 ≤ 0.4) and a Br2 yield on the order of 50 % are observed on non-reactive salts with a closed mass balance for Br2. The ClONO2-salt system has been reinvestigated. Experiments on NaCl, KBr (reactive salts) and on NaNO3 and Na2SO4 (non-reactive salts) have been performed. The hydrolysis of ClONO2 was not observable in contrast to heterogeneous decomposition which occurred on non-reactive salts with a Cl2 yield of (25 ± 5) %. Calculations have been performed using a 0-D box model in order to observe the effects of heterogeneous chemistry on sea salt in the marine boundary layer. Chlorine and bromine chemistry is activated by heterogeneous reactions occurring on NaCl and KBr which are the sole sources of chlorine and bromine in the model. Simulations performed with chlorine heterogeneous chemistry and gas phase chemistry indicate: 1) Denitrification of the troposphere (NOy converted to solid nitrates). 2) Increase of the oxidative capacity of the troposphere by the release of Cl atoms. When bromine chemistry is added to the model, the recombination reaction of NO2 with BrO leads to an increase of tropospheric denitrification through formation of BrONO2. Calculations show that at high [NOx], the most important bromine reservoir is BrONO2, while at low [NOx] HOBr is the most important bromine reservoir. The uptake of HNO3 on ice, solid sulfuric acid solutions and solid ternary solutions (STS) has been investigated at 180 to 211 K. The uptake coefficient γ decreases from 0.30 at 180 K to 0.06 at 211 K. The Arrhenius representation shows two distinct regimes. The first at low temperatures (180-190 K) shows a constant value γ of 0.3, whereas the second regime at T > 195 K corresponds to an activation energy of Ea = -7 ± 1 kcal/mol. At a fixed temperature, γ is independent of [HNO3] thus confirming a rate law first order in [HNO3]. The γ values of HNO3 on H2SO4/H2O solid solutions linearly decreases from 0.20 (0.10) at 10 wt % H2SO4 to 0.05 (0.03) at 98 wt % H2SO4 at 180 K (200K). The γ values of HNO3 with STS of H2SO4/HNO3/H2O is equal to 0.10 ± 0.03 in the temperature range 185-195 K at conditions where the composition of the interface was held constant at the given temperature by adding an external flow of H2O. Br2O hydrolysis on ice surfaces occurs rapidly in the chosen temperature range of 180 to 210 K. At a fixed temperature the uptake kinetics follow an apparent first order rate law in [Br2O]. The observed negative temperature dependence leads to an activation energy Ea for heterogeneous hydrolysis of -2.3 ± 0.6 kcal/mol. These facts point towards a complex reaction mechanism implying that the interaction of Br2O with ice is not an elementary reaction. BrONO2 hydrolysis has been measured on bulk (B), condensed (C) and single crystal (SC) ice in the temperature range 180-210 K. On all types of ice, HOBr and Br2O are observed as products. At a fixed temperature the rate law is first order in [BrONO2] with γ ≈ 0.3 at 180 K. The observed negative temperature dependence leads to an activation energy Ea for the hydrolysis of BrONO2 on pure ice of -2.0 ± 0.2, -2.1 ± 0.2 and -6.6 ± 0.3 kcal/mol on (C), (B) and (SC) ice, respectively, pointing towards different kinetics of BrONO2 on these different types of ice. Doping the ice samples with HBr leads to the formation of Br2, preceded by BrONO2 hydrolysis. The Arrhenius representation corresponds to an activation energy of Ea = -1.2 ± 0.2 kcal/mol. We investigated the structural properties of the near-surface region of various types of HX (X = Cl or Br) doped ice. Basically, we studied the time dependent [HX] in the interface region using the fast reaction titration XONO2 + HX → X2 + HNO3. These experiments reveal that HX is located near the surface of the substrate in a well defined region of thickness I. The HX molecules composing the interface are immediately available for the titration reaction, whatever the flow of XONO2. In the temperature range 190-200 K, we measured a value of IHCl = 43 ± 16, 250 ± 50 and 444 ± 120 nm for (SC), (C) and (B) ices, respectively and IHBr = 110 ± 15 nm on (C) ice at 205K. Finally, we assessed the bulk diffusion coefficient for HX, DHX by modeling our results according to Fick's laws of diffusion. We obtained values of DHCl = 4.5·10-15 - 1.0·10-12 cm2/s at 190 K and DHBr = (6.5 ± 3.0)·10-15 cm2/s at 205 K.

EPFL2001

Kinetic modeling of dissolution and crystallization of batch reactions with in situ spectroscopic measurements

Julien Léo Billeter

The use of Process Analytical Technologies (PATs) recommended by the Food and Drug Administration (FDA) has significantly increased during the past years in the design, control and monitoring of pharmaceutical or chemical manufacturing processes [1]. Nowadays PAT is also commonly used in Good Manufacturing Practices (GMPs). Some PAT techniques employ on-line fiber-optic sensors to acquire non-destructive measurements of physical properties and kinetic information of dissolved and solid fraction of molecular substances in slurries in real time [2]. The main objective of this project is to develop a global model for DuPont’s sulfonylurea coupling reaction for monitoring purposes (e.g. detect process upset, detect endpoint, and forecast changes). The global model will allow us to estimate the kinetics of the reaction, the kinetics of nucleation, the kinetics of dissolution, and the kinetics of crystallization. Before such complex model can be developed, it is necessary to conduct experiments in a simpler system (e.g. salicylic acid in water-ethanol mixture). These experiments allow us to control critical parameters and establish precisely known conditions. In addition, they are designed so the process of dissolution and the process of crystallization can be observed independently one another, and independently of chemical reactions. Consequently, we will be able to develop an appropriate model for each process in our small-scale semi-batch reactor. For the simpler system, this study uses attenuated total reflectance ultra-violet visible (ATR UV-vis) spectroscopy for kinetic modeling of the dissolution and crystallization of salicylic acid in ethanol-water. The dissolution model, which relies on a power-law equation, can be obtained by adding aliquots of an ethanol-water mixture into a salicylic acid slurry. Near-infrared (NIR) diffuse reflectance spectroscopy is used to detect the degree of saturation, quantify the solid fraction present in a slurry using a calibration, and for verification and validation purposes. A temperature probe is also used to monitor heat changes involved in this dissolution. [1] Fevotte G., International Journal of Pharmaceutics 241 (2002) 263-278 [2] Gemperline et al, Analytical Chemistry 76 (2004) 2575-2582

2010

Investigation of Biophysical Properties of Biomolecules by Means of Atomic Force Microscopy

Jae Sun Jeong

Biomolecules are the building blocks of living organisms and perform the most important functions in a biological process. The aim of biophysics is to understand the biological functions of biomolecules in terms of their structure: structure and function of biomolecules has been an active research for several decades. Despite the evolution and emergence of new investigation techniques during the last 60 years, biological processes still present enormous challenges to our understanding due to the complex biological system spanning a wide range of spatial and temporal scales. An ideal biophysical method should have the capability to observe atomic level structures and dynamics of biological molecules in their physiological environment. It would also permit the visualization of the structures that form throughout the course of conformational changes or chemical reactions, regardless of the time scale. In this thesis, we have investigated two representative biomolecules using the well-defined biophysical methods in terms of behaviour and biophysical properties of biomolecules: Deoxyribonucleic acid (DNA) and the Amyloid-β (Aβ) peptide. Many different biophysical and biochemical tools were employed, where atomic force microscopy (AFM) was the primary instrument to observe the behaviour of the biomolecules in physiological conditions as well as the dynamic changes of structure of biomolecules. Moreover, AFM was used to measure the mechanical properties of biomolecules via direct and indirect methods. What is the behaviour of DNA molecules in an electric field? The behaviour of DNA molecules in an electric field is crucial for understanding interactions of DNA with electrically charged membrane that serves as a template for DNA based sensors and microarray applications, particularly under an electric field. Taken together, the ability to deposit a single DNA molecule on the electrode is essential to increase its specificity. To achieve this, we optimized the operational conditions in order to control a single DNA molecule adsorbing at +300 mV of potential and desorbing to/from the electrode at -25 mV of potential. Simultaneously dynamic imaging enabled us to analyse its morphological behaviour using real-time Electrochemical AFM (EC-AFM) in aqueous conditions. What is the molecular mechanism underlying Aβ42-fibrillogenesis and its role in pathogenesis? The molecular mechanism underlying Aβ42-fibrillogenesis is essential for defining the key determinant in the pathogenesis of Alzheimer’s disease. In this thesis, we carried out detailed biophysical studies to elucidate the structural changes associated with different stages of amyloid formation and provided unique perspectives on the molecular and structural basis underling the polymorphism of amyloid fibrils in Alzheimer’s disease. For the first time, we provided direct evidence of the existence of secondary-nucleation sites on the surfaces of Aβ42-fibrils and demonstrated that Aβ42-monomers play a crucial role in determining the structural properties and heterogeneity of the formed fibrils. In addition, real-time observation with in situ AFM was performed to define the mechanisms underlying oligomerization and the formation of protofibrils of Aβ42 in physiological conditions. Investigation of the elastic property of Aβ42 fibrils using two techniques based on the AFM. The self-assembly process of Aβ peptides into highly ordered amyloid fibrils is a crucial phenomenon in the cascade of pathological events that culminates in Alzheimer’s disease. Further, the assessment of the elastic properties of Aβ fibrils has recently attracted a lot of attention due to their potential biological applications. We measured the elastic property of Aβ42-fibrils immobilized on two types of substrate (positively charged mica and hydrophobic highly oriented pyrolytic graphite) and compared their properties using two techniques based on the AFM: Statistical analysis of thermal fluctuations and AFM based nanoindentation. The results from our studies show that the values of Young’s modulus of Aβ42 fibrils from the two substrates were similar (1.8 to 2.0 GPa), however they were different in the statistical analysis of thermal fluctuations (1.4 GPa, 2.3 GPa on mica and highly oriented pyrolytic graphite, respectively). These results imply that the method of thermal fluctuations was based on the shape of fibrils affected by the interaction force between the substrate and the fibrils.

EPFL2012