Fluorescence relaxation spectroscopy : light on dynamical structures of flavoproteins

Refinements in technique and data analysis have opened new avenues for a detailed interpretation of protein fluorescence. What is more, by combining new insights in protein structure and dynamics with improved knowledge of photophysics of biological chromophores, the coupling between structure-function relationships and fluorescence properties is beginning to have a firm foundation. As a consequence of this interdisciplinary marriage, bidirectional information can be inferred from fluorescence spectroscopy of proteins.In chapter 2 we could explain the time-resolved fluorescence parameters of FAD in glutathione reductase and lipoamide dehydrogenase by a different interplay between conformational substates in both enzymes. The existence of these states monitored by fluorescence relaxation spectroscopy, allowed us to postulate a new model of substrate binding prior to catalysis (Fig.1).In this model, the majority of enzyme molecules in solution is present in an inaccessible conformation to substrate or cofactor ("closed" conformation, A in Fig.1). Structural (equilibrium) fluctuations give rise to a minor population of enzyme molecules with a proper conformation to bind substrate (" open " conformation, B in Fig.1). The equilibrium between "open" and "closed" enzyme conformations is shifted towards the "open" form in the presence of excess substrate or cofactor molecules (C in Fig.1) which reduce the flavin on the enzyme (D in Fig.1). The structural transition can be the rate limiting step in catalysis if the activation barrier between the "open" and "closed" enzyme structures is sufficiently large.From time-resolved fluorescence anisotropy measurements as function of temperature one can distinguish between reorientational motion and energy transfer. In both dimeric proteins intersubunit energy transfer takes place between the FAD prosthetic groups. Furthermore, restricted reorientational motion of the flavin is only revealed in glutathione reductase. It is very likely that this flexible isoalloxazine is present in a minor population of enzyme molecules with a displaced Tyr197 making the flavin accessible to the cofactor (NADPH). This catalytically important enzyme species is not detected by X-ray crystallography and its observation requires a technique with high dynamic range of intensity such as time-resolved fluorescence. In order to derive the distance and relative orientation between the flavins, separate models describing fluorescence anisotropy must be applied to both proteins. The geometric arrangement of the flavins as derived from the analysis of the results is in excellent agreement with crystallographic data.Chapter 3 of this thesis deals with the dynamic properties of the flavin environment in Azotobacter vinelandii lipoamide dehydrogenase as determined by steady-state fluorescence spectroscopy. As model systems the native enzyme and a deletion mutant lacking the 14 C-terminal amino acids were chosen. By fluorescence quenching and temperature dependent spectroscopy it was shown that the mutant has a flavin site accessible to solvent in contrast to wild type lipoamide dehydrogenase. We have to conclude from these observations and enzyme kinetic studies [1] that the C-terminal polypeptide of lipoamide-dehydrogenase is folded back onto the dehydrolipoamide binding site. This result is in agreement with the crystal structure of the Pseudomonas putida enzyme (A. Mattevi, personal communication) but in discrepancy with the disordered C-terminal tail of crystalline A.vinelandii lipoamide dehydrogenase [2]. In cryogenic solvents different dipolar relaxation behavior was found for mutant and native enzymes. The flavin environment in the wild type enzyme was rigid on a nanosecond timescale while a conversion from slow to rapid relaxation was observed in the mutant protein from 203 to 303 K. The contributions to dipolar relaxation in the mutant enzyme were found to arise from solvent and protein dipoles. In aqueous solution, rapid relaxation was observed in both proteins indicating that rapid protein fluctuations are damped in viscogens. Because of the difference in dipolar relaxation in wild type and mutant proteins, the unique spatial arrangement of the two flavins makes this biological system an appropriate object to study the relationships between homo-energy transfer, dipolar relaxation and excitation energy. In this context a compartmental theoretical model is developed in chapter 4 on the basis of existing qualitative descriptions [3, 4]. The model was verified by global analysis of fluorescence anisotropy decay surfaces. The geometry and dynamics derived from the results were in excellent agreement with the protein-crystal geometry [2] and with the dipolar relaxation elaborated in chapter 3. In addition, thermodynamic parameters of equilibrium fluctuations in the protein structures could be determined from the maximum entropy analysis of the fluorescence decay. In agreement with Frauenfelder's concept of tiers of conformational substates [5, 6], the existence of two classes of states were established in lipoamide dehydrogenase (CS 1and CS 3). Thermal quenching of flavin fluorescence was related to transitions between conformational substates of category CS 3by comparing activation energy values.In chapter 5 red- and blue-edge spectroscopy was applied to the electron-transferring flavoprotein from Megasphaera elsdenii which contains two FAD molecules within one subunit. Energy transfer between the two flavins was observed from timeresolved fluorescence depolarization experiments. We could demonstrate for the first time that energy transfer is not observable (as theoretically predicted) at the blue-edge of the emission band in an inhomogeneously broadened system. Interflavin distance and the relative orientation of the flavins could be determined for this protein with unknown threedimensional structure.In general, owing to a physical model based on conformational substates in proteins it is possible to use the complex fluorescence decay patterns in enzymes to explain certain aspects of catalysis. The analysis of the fluorescence decay in distributions of lifetimes by the maximum entropy method is then the logical choice since the information on conformational states is contained in the multiplicity of the distribution pattern. The advantage of the maximum entropy method is that no a priori knowledge of the complex protein system needs to be encoded. According to the fluctuation-dissipation theorema [5] it is possible to monitor catalytically important protein conformations without substrate present. In order to test this hypothesis, fluorescence studies of binary enzyme-substrate complexes must be undertaken in the future. Catalytically important conformational substates will then be preferentially populated in contrast to the free enzyme.We have also demonstrated that the validity of physical models on energy transfer in relation to dipolar relaxation can be tested in relatively complex biological systems. Precise goniometric information in bichromophoric proteins can be derived from fluorescence anisotropy relaxation. Temperature and excitation energy-dependent studies of the fluorescence anisotropy provide a diagnostic method to distinguish reorientational motion from energy transfer. The described experimental and analytical methods have broad applicability to other biological systems containing identical fluorophores

Fluorescence relaxation spectroscopy : light on dynamical structures of flavoproteins

Refinements in technique and data analysis have opened new avenues for a detailed interpretation of protein fluorescence. What is more, by combining new insights in protein structure and dynamics with improved knowledge of photophysics of biological chromophores, the coupling between structure-function relationships and fluorescence properties is beginning to have a firm foundation. As a consequence of this interdisciplinary marriage, bidirectional information can be inferred from fluorescence spectroscopy of proteins.In chapter 2 we could explain the time-resolved fluorescence parameters of FAD in glutathione reductase and lipoamide dehydrogenase by a different interplay between conformational substates in both enzymes. The existence of these states monitored by fluorescence relaxation spectroscopy, allowed us to postulate a new model of substrate binding prior to catalysis (Fig.1).In this model, the majority of enzyme molecules in solution is present in an inaccessible conformation to substrate or cofactor ("closed" conformation, A in Fig.1). Structural (equilibrium) fluctuations give rise to a minor population of enzyme molecules with a proper conformation to bind substrate (" open " conformation, B in Fig.1). The equilibrium between "open" and "closed" enzyme conformations is shifted towards the "open" form in the presence of excess substrate or cofactor molecules (C in Fig.1) which reduce the flavin on the enzyme (D in Fig.1). The structural transition can be the rate limiting step in catalysis if the activation barrier between the "open" and "closed" enzyme structures is sufficiently large.From time-resolved fluorescence anisotropy measurements as function of temperature one can distinguish between reorientational motion and energy transfer. In both dimeric proteins intersubunit energy transfer takes place between the FAD prosthetic groups. Furthermore, restricted reorientational motion of the flavin is only revealed in glutathione reductase. It is very likely that this flexible isoalloxazine is present in a minor population of enzyme molecules with a displaced Tyr197 making the flavin accessible to the cofactor (NADPH). This catalytically important enzyme species is not detected by X-ray crystallography and its observation requires a technique with high dynamic range of intensity such as time-resolved fluorescence. In order to derive the distance and relative orientation between the flavins, separate models describing fluorescence anisotropy must be applied to both proteins. The geometric arrangement of the flavins as derived from the analysis of the results is in excellent agreement with crystallographic data.Chapter 3 of this thesis deals with the dynamic properties of the flavin environment in Azotobacter vinelandii lipoamide dehydrogenase as determined by steady-state fluorescence spectroscopy. As model systems the native enzyme and a deletion mutant lacking the 14 C-terminal amino acids were chosen. By fluorescence quenching and temperature dependent spectroscopy it was shown that the mutant has a flavin site accessible to solvent in contrast to wild type lipoamide dehydrogenase. We have to conclude from these observations and enzyme kinetic studies [1] that the C-terminal polypeptide of lipoamide-dehydrogenase is folded back onto the dehydrolipoamide binding site. This result is in agreement with the crystal structure of the Pseudomonas putida enzyme (A. Mattevi, personal communication) but in discrepancy with the disordered C-terminal tail of crystalline A.vinelandii lipoamide dehydrogenase [2]. In cryogenic solvents different dipolar relaxation behavior was found for mutant and native enzymes. The flavin environment in the wild type enzyme was rigid on a nanosecond timescale while a conversion from slow to rapid relaxation was observed in the mutant protein from 203 to 303 K. The contributions to dipolar relaxation in the mutant enzyme were found to arise from solvent and protein dipoles. In aqueous solution, rapid relaxation was observed in both proteins indicating that rapid protein fluctuations are damped in viscogens. Because of the difference in dipolar relaxation in wild type and mutant proteins, the unique spatial arrangement of the two flavins makes this biological system an appropriate object to study the relationships between homo-energy transfer, dipolar relaxation and excitation energy. In this context a compartmental theoretical model is developed in chapter 4 on the basis of existing qualitative descriptions [3, 4]. The model was verified by global analysis of fluorescence anisotropy decay surfaces. The geometry and dynamics derived from the results were in excellent agreement with the protein-crystal geometry [2] and with the dipolar relaxation elaborated in chapter 3. In addition, thermodynamic parameters of equilibrium fluctuations in the protein structures could be determined from the maximum entropy analysis of the fluorescence decay. In agreement with Frauenfelder's concept of tiers of conformational substates [5, 6], the existence of two classes of states were established in lipoamide dehydrogenase (CS 1and CS 3). Thermal quenching of flavin fluorescence was related to transitions between conformational substates of category CS 3by comparing activation energy values.In chapter 5 red- and blue-edge spectroscopy was applied to the electron-transferring flavoprotein from Megasphaera elsdenii which contains two FAD molecules within one subunit. Energy transfer between the two flavins was observed from timeresolved fluorescence depolarization experiments. We could demonstrate for the first time that energy transfer is not observable (as theoretically predicted) at the blue-edge of the emission band in an inhomogeneously broadened system. Interflavin distance and the relative orientation of the flavins could be determined for this protein with unknown threedimensional structure.In general, owing to a physical model based on conformational substates in proteins it is possible to use the complex fluorescence decay patterns in enzymes to explain certain aspects of catalysis. The analysis of the fluorescence decay in distributions of lifetimes by the maximum entropy method is then the logical choice since the information on conformational states is contained in the multiplicity of the distribution pattern. The advantage of the maximum entropy method is that no a priori knowledge of the complex protein system needs to be encoded. According to the fluctuation-dissipation theorema [5] it is possible to monitor catalytically important protein conformations without substrate present. In order to test this hypothesis, fluorescence studies of binary enzyme-substrate complexes must be undertaken in the future. Catalytically important conformational substates will then be preferentially populated in contrast to the free enzyme.We have also demonstrated that the validity of physical models on energy transfer in relation to dipolar relaxation can be tested in relatively complex biological systems. Precise goniometric information in bichromophoric proteins can be derived from fluorescence anisotropy relaxation. Temperature and excitation energy-dependent studies of the fluorescence anisotropy provide a diagnostic method to distinguish reorientational motion from energy transfer. The described experimental and analytical methods have broad applicability to other biological systems containing identical fluorophores

Imaging protein interactions by FRET microscopy: FRET measurements by acceptor photobleaching

This protocol describes the detection of fluorescence resonance energy transfer (FRET) by measuring the quenching of donor emission alone. As opposed to sensitized emission measurements, photobleaching can be performed with high selectivity of the acceptor because absorption spectra are steep at their red edge, allowing the acceptor to be bleached without excitation of the donor. When using acceptor photobleaching FRET measurements, care should be taken that the photochemical product of the bleached acceptor does not have residual absorption at the donor emission and, more importantly, that it does not fluoresce in the donor spectral region. Because of mass movement of protein during the extended time required for photobleaching (typically 1-20 min), it is preferable to perform this type of FRET determination on fixed cell samples. Live-cell FRET measurements based only on donor fluorescence are more feasible using fluorescence lifetime imaging (FLIM), because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor

Imaging protein interactions by FRET microscopy: FRET measurements by sensitized emission

This protocol describes a method for measuring fluorescence resonance energy transfer (FRET) by the detection of acceptor-sensitized emission. This approach is useful in situations where donor intensities are low and/or there is contamination with high background (auto) fluorescence in the donor channel. However, absorption spectra characteristically exhibit long tails in the higher-energy, shorter-wavelength (blue) region, which may result in the direct excitation of the acceptor molecule in addition to that of the donor, thus resulting in mixing of direct and sensitized emission. Conversely, fluorescence emission tends to tail into the red part of the spectrum, causing donor fluorescence bleed-through into the acceptor detection channel. Corrections for these effects involve the acquisition of fluorescence images of samples containing the donor, the acceptor, and both of these for three different filter settings. The result is an estimation of the sensitized emission, i.e., the emission induced by FRET from the donor to the acceptor alone. Excitation of a donor fluorophore in a FRET pair leads to quenching of the donor fluorescence and increased emission from the acceptor (sensitized emission). This can be normalized using the acceptor emission, measured after specific excitation of the acceptor, to define apparent energy transfer efficiency in each pixel of the image. It is also proportional to the fraction of acceptor molecules that is bound to a donor-tagged molecule. Alternatively, an apparent energy transfer efficiency can also be defined that is proportional to the bound fraction of donor-tagged molecules

Imaging protein interactions by FRET microscopy: cell preparation for FRET analysis

The following protocol describes the preparation of cells for FRET analysis on live and fixed cells. The reagents used have been optimized to minimize the quenching of GFP mutants and fluorescent dyes