oxygen transfer proteins and redox enzymes
Zauner, G.
Citation
Zauner, G. (2008, October 23). A FRET-based method to study the activity of electron or oxygen transfer proteins and redox enzymes. Retrieved from https://hdl.handle.net/1887/13201
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Chapter VII
General discussion, conclusions and
perspective
General discussion, conclusions and perspective
This thesis centers on a novel application of Förster Resonance Energy Transfer (FRET) (1): we use a fluorescent dye molecule covalently attached to the protein as the FRET donor (2-7). The intrinsic redox-co-factor of this protein functions as the energy acceptor that is assumed to be in a constant distance to the attached dye molecule of which the fluorescence intensity is monitored. Therefore not the conventional ‘spectroscopic ruler’ approach (8) is applied where the change in the distance between a donor and an acceptor molecule is measured. Our technique takes advantage of the fact that the optical characteristics of the cofactor may vary (1) upon change in redox state or (2) upon binding of a ligand/substrate to the active site. This change in the absorption spectrum of the protein’s cofactor translates into a change in the fluorescence intensity of a fluorophore by means of FRET. In this way, the distance between donor and acceptor remains constant, whereas changes in the energy transfer efficiency (R0) due to differences in acceptor absorption are observed.
In the studies described in this thesis the redox state of the protein or the substrate binding to the prosthetic group of an enzyme was, thus, monitored by a much more sensitive and selective approach than conventional absorption measurements. The technique used for all the studies was fluorescence spectroscopy. The main aim of the thesis work was to investigate the applicability of our FRET based approach to first of all various proteins/enzymes harbouring different prosthetic groups. Secondly, we demonstrated that it is even feasible to investigate the enzymatic turnover of single surface-confined enzyme molecules labeled with a fluorophore using scanning confocal fluorescence microscopy. The approach is highly valuable as the method provides us with a sensitive tool to monitor biological redox events (2;7). In principle any redox protein which shows a redox dependent change in its absorption spectrum can be addressed with our method. The observation of single labeled proteins opens the door to the world of single enzymatic studies, a rather new field full of technical
General discussion, conclusions and perspective
challenges (9). These single enzyme studies have been shown to unravel information that is hidden in ensemble-averaged measurements (10;11).
Methods to determine the protein’s redox state
To study the mechanism of redox enzymes and the function of electron transfer chains it is important to be able to monitor the redox state of the prosthetic groups (3). Normally conventional redox titrations employ the absorption spectrum of the protein to relate back to the redox potential in order to determine the midpoint potential of a redox protein. This approach may become problematic when the protein concentration is low or when the sample contains several components with overlapping absorption spectra.
We could employ our FRET based method for sensing the changes in protein absorption spectra by means of an attached dye molecule (2;3). Three different prosthetic groups have been used to demonstrate the wide applicability of the process as shown in Chapter II. The method permits to reliably distinguish between reduced and oxidized proteins and to perform potentiometric titrations by following the fluorescence intensity of the attached dye molecule. The biggest advantages of our approach are (1) the low concentration of protein required, (2) the selectivity as the attached fluorophore mostly emits in a region clearly distinguishable from other absorbing/emitting substances and (3) the short timescale of the energy transfer, meaning that there are no delays in the responses observed.
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Methods to monitor substrate binding to the active site of the protein
When monitoring a ligand binding event to a protein/enzyme for most cases a spectroscopic method is applied. Observing changes in the protein absorption upon substrate binding and/or turnover can possibly become problematic due to interferences with other substances in a turbid (biological) solution or due to the absorbance of the ligand itself which might play a role. The measure of the intrinsic tryptophan fluorescence of several proteins has been shown to be useful for monitoring ligand binding too (1;12;13) even though background fluorescence in the near UV is unavoidable especially in biological samples.
We could circumvent the above mentioned problems by applying our FRET based approach in an ‘extended’ form: A scheme for oxygen sensing in solution based on type 3 copper proteins was developed (Chapter III). The method exploits the sensitivity of the endogenous fluorescence of type-3 copper proteins towards the presence of oxygen by translating the near-UV emission of the protein to label fluorescence in the visible range through a FRET mechanism. This solution method provides us again with a biocompatible and rather selective tool in terms of specificity as even two or more conjugates can be monitored at the same time. Additionally it allows a ‘freedom of choice’ of the fluorescent dye molecule to tune the emission wavelength to specific applications. In terms of sensor response time measurements down to the millisecond timescale are feasible. Coming back to the above mentioned intrinsic tryptophan fluorescence, which is decreasing upon binding of oxygen to the active site of type 3 copper proteins, it could be shown that the label contrast between O2-free and O2-bound protein can be made to exceed the contrast observed for the tryptophan fluorescence. Combining all these advantages with fluorescence microscopy methods would possibly enable measurement of oxygen consumption at sub-cellular levels and millisecond timescales and to monitor, for example, metabolic
General discussion, conclusions and perspective
activity. Although the method has been described for its application in oxygen sensing, it can be utilized, in principle, for all systems displaying tryptophan fluorescence changes.
In addition, a solid state sensor based on the same approach could be developed (Chapter IV). In Chapter IV we describe how the principle introduced in Chapter III can be adapted to sense O2 by means of type 3 copper proteins immobilized in optically transparent silica matrices. The two compounds used for protein encapsulation are Alkoxide TetraMethOxySilane (TMOS) and Sodium metasilicate (Na2SiO3), both of which are well studied and appeared to be excellent for applications in biosensors. It has been shown that the device features a fast response, biocompatibility, reusability and outstanding stability.
In Chapter V we describe the application of the FRET principle to P450cam from Pseudomonas putida to monitor substrate binding to the active site of the protein. The fluorescence intensities of the attached label could be used to determine the dissociation constants for two different substrates, a high and a low affinity one. The values obtained from fluorescence measurements match the data acquired by absorption spectroscopy with the main advantage of requiring at least ten times less enzyme. Again the wavelength for the emission read out is advantageous compared to absorption measurements or the measure of the intrinsic tryptophan emission in a turbid solution.
Methods to monitor enzyme activity at the single molecule level
Currently existing single-molecule enzymatic assays based on fluorescence are limited to the flavoenzymes, which contain a fluorescent cofactor (14;15), and to the enzymes for which a fluorogenic substrate can be designed (11;16;17). Our method could be
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shown to have promise for single enzyme studies (Chapter VI). From the fluorescence time traces of a labeled enzyme (copper-containing nitrite reductase from Alcaligenes faecalis S-6), monitored under turn-over conditions, the kinetic parameters could be extracted and connected to the macroscopic ensemble averaged kinetic constants. The fact that it is not directly based on fluorescence as most other studies, but on the absorption properties of an enzyme’s cofactor translated into fluorescence, the method described in our work can dramatically increase the range of redox enzymes, for which the kinetics can be studied by single molecule fluorescence methods. It can be potentially applied to any enzyme for which the absorption significantly changes during its catalytic cycle.
Conclusions and future perspectives
Our FRET based approach seems auspicious for many applications as it is broadly applicable and has the following advantages:
- The time resolution of the technique is only limited by the time scale of the Förster energy transfer, which amounts up to nsec (18);
- The sensitivity of fluorescence measurements allows even to go down to the single molecule level enabling single enzyme studies by means of FRET via an attached dye molecule;
- By using fluorophores emitting at a specific wavelength the method permits to exclusively monitor ‘protein events’ in a specific way even in biological sample solutions;
- The ‘sensitized fluorescence’ approach introduced in Chapter III allows to tune the wavelength for the read out to the one required for detection;
General discussion, conclusions and perspective
- The broad application in itself is a big advantage of the method as in principle it can be applied to any redox protein/enzyme exhibiting a change of absorption spectrum upon a certain event;
- Moreover, for enzymes containing more than one redox cofactor with different absorption spectra, labeling with different fluorescent dyes reporting on the redox state of the individual cofactors, would provide a deeper insight into the enzyme mechanism first of all in bulk and potentially at the single- molecule level. Also monitoring protein-protein interactions from both sides, i.e. following the electron transfer from one to the other protein by means of FRET should be viable. In that case the selected fluorophores should be chosen in a way to not transfer energy between themselves but only to the prostethic group of the protein.
For future perspectives at least two goals seem worthwhile to pursue:
- First of all, out of scientific curiosity the kinetics of an enzyme, not characterized yet by ensemble measurements should be unravelled to demonstrate the outstanding qualities of the method.
- Second, to find a biosensing application based on the fluorescence method possibly in combination with the immobilization of an enzyme on a surface (Chapter VI) or into a matrix (Chapter IV), with the implementation of quantum dots as the FRET donors (19) or anchoring the enzyme for a spectroelectrochemical approach (20) should be tried. Considering all the efforts made so far, a combination of several techniques seems feasible. The beauty in our case is that the method developed here allows a more or less free choice of (redox) enzyme to be studied.
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General discussion, conclusions and perspective
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