Retardation of Bulk Water Dynamics by Disaccharide Osmolytes

The bioprotective nature of disaccharides is hypothesized to derive from the modification of the hydrogen bonding network of water which protects biomolecules through lowered water activity at the protein interface. Using ultrafast fluorescence spectroscopy, we measured the relaxation of bulk water dynamics around the induced dipole moment of two fluorescent probes (Lucifer Yellow Ethylenediamine. and Tryptophan). Our results indicate a reduction in bulk water reorganization rate of approximately 30%. We observe this retardation in the, low concentration regime measured at 0.1 and 0.25 M, far below the onset of glassy dynamics. This reduction in water activity could be significant. in crowded biological systems, contributing to global change in protein energy landscape, resulting in a significant enhancement of protein stability under environmental stress. We observed similar dynamic reduction for two disaccharide osmolytes, sucrose and trehalose, with trehalose being the more effective in reducing solvation dynamics.

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

Operation of Complex Hydropower Schemes and its Impact on the Flow Regime in the Downstream River System under Changing Scenarios

Martin Peter Bieri

Hydropower is the world’s most important renewable electricity source. More than 40% of European hydroelectric energy is produced in Alpine countries. High-head storage hydropower plants (HPP) contribute significantly to peak energy production as well as electricity grid regulation. Future plant management is faced with several challenges concerning modified availability of water resources due to climate change as well as new economic constraints associated with legal, political and electricity market issues. HPP operation results in unsteady water release to the downstream river system. Hydropeaking is the primary factor of flow regime alteration, impacting the river ecosystem. Even when the biological response to hydropeaking is not fully understood, the recently adapted law on water protection prescribes its mitigation in Switzerland. In this research project, a novel integrative approach to model and assess the impact of the operation of a complex hydropower scheme on the downstream river system is developed. It contains (1) a precipitation-runoff model extended for long-term simulations of glacierized Alpine catchment areas, (2) an operation tool for high-head storage HPP, (3) flow regime generation with cost estimation of hydropeaking mitigation measures and (4) a habitat model of reference river morphologies for a target species. The upper Aare River (Hasliaare) in Switzerland is an Alpine stream, affected by hydropeaking from a complex hydropower scheme with several storage volumes and power houses. Since the 1930s, seasonal water transfer from summer to winter and the amplitude and frequency of daily peak discharge have been continuously increased. Furthermore, the dynamic braided river network with various mesohabitats gave way to a mainly monotonous channel. Although diversity of species and biomass of aquatic biota have drastically decreased, the potential of redevelopment remains. Investigations to improve the river morphology and the flow regime are under discussion. The upper Aare River catchment is therefore an appropriate case study for analysis of the interactions between climatic, hydrological, hydraulic, economic as well as ecological parameters. The simulation of runoff in Alpine catchment areas is essential for optimal hydropower exploitation under normal flow conditions, but also for the analysis of flood events. The semi-distributed conceptual modeling approach Routing System contains a reservoir-based precipitation-runoff transformation model (GSM-SOCONT), extended by dynamic glacier simulation tool. Spatial precipitation and temperature distributions are taken into account for simulating the relevant hydrological processes, such as glacier melt, snowpack constitution and melt, soil infiltration and runoff. The model development, calibration and validation are illustrated for the 2005 flood event, where the flood reduction capacity of the HPP is discussed, as well as future long-term runoff estimations. Climate change scenarios, based on a reference climate period, take into account intra-annual temperature and precipitation variations as well as their long-term tendencies. Runoff series of daily resolution are produced by hourly updating of the meteorological, glaciological and hydrological parameters. An almost complete deglacierization of the upper Aare River basin is simulated for the late 21st century. The resulting reduction of glacier melt in summer and earlier snowmelt in spring change the runoff regime from glacio-nival to nival. The implemented heuristic hydropower modeling tool in Routing System allows simulation of the operating mode of complex HPP. Within the case study of the upper Aare River catchment and despite the complexity of the HPP network, the influence of climate change, electricity market issues, plant enhancements as well as hydropeaking constraints is simulated and assessed. Despite the reduction of future runoff, increased flexibility due to new turbine and pumped-storage capacities allows compensation, especially in the case of volatile electricity prices, and could even partially restore the natural flow regime. Several operational and construction measures to reduce hydropeaking are implemented in the model. Resulting flow regimes as well as the related costs are defined. Operational constraints, such as limitation of turbine discharge, increase of residual flow or limited drawdown range, generate relatively high costs compared to their environmental effectiveness. Better ecological and economic response is achieved by construction measures, such as flow deviation systems or compensation basins installed downstream of the power house outflow where the water is temporarily stored and then released to the river by a guided system. The simulated flow regimes are rated by a river specific habitat model for representative morphologies and three life stages of the target species brown trout (Salmo trutta fario). This is based on results from a 2D hydrodynamic model and in situ investigations undertaken in the framework of a joint project of EAWAG. Steady and dynamic indicators quantify fish habitat suitability and allow comparison through economic indices of the implemented mitigation measures. For the Hasliaare River, investments for mitigation of hydropeaking are only justified by morphological improvements. The developed approach is useful for the enhancement of complex storage hydropower schemes regarding mitigation of altered flow regimes. Despite several uncertainties, it allows operators, authorities and researchers to define and rate the impact of HPP operation on the river network, to ecologically and economically assess mitigation measures and thus to address hydropeaking in a straightforward manner.

EPFL2012

Operation of complex hydropower schemes and its impact on the flow regime in the downstream river system under changing scenarios

Martin Peter Bieri

EPFL - LCH2012

Operation of Complex Hydropower Schemes and its Impact on the Flow Regime in the Downstream River System under Changing Scenarios

Martin Peter Bieri

EPFL2012

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

Selma Schenkl

EPFL2004

Operation of complex hydropower schemes and its impact on the flow regime in the downstream river system under changing scenarios

Martin Peter Bieri

EPFL - LCH2012