Creation of a novel signal transducer using a set of electron transfer proteins
Enzyme-based biosensors exhibit high sensitivity to a specific substance as they use the ability of substrate recognition of the enzymes. Owing to the high compatibility to a living body, active use and research have been made in various fields (Ref. 1). Sensors consist of two parts; a sensing site that senses a change in the environment and the presence of some substance to generate a primary signal and a signal transduction site that converts the primary signal to a secondary one readily detectable and processible. Because of easy handling as data, electrical signal is widely used as the secondary signal. This point is also true of the biosensor using enzymes. However, since there is no signal transduction system in the enzymes, the primary signal (ie enzymatic reaction) should be electronic or electrically detectable in many biosensors available. This is one of the major reasons of restricted usability of the biosensors. Meanwhile, in natural sensor proteins, a primary signal caused by a sensing event (some reaction) induces a conformational change of protein, which then, generates a secondary signal. Thus, the conformational change of the proteins serves as signal transducing mechanism (Figure 1). Artificial reproduction of such a sophisticated system should be a target to create a biosensor having a signal transducing mechanism, which would expand the application range of the biosensor. However, it is highly challenging yet to design such mechanism at the molecular level using a single protein (Ref. 2). In order to solve this problem, we attempted to replace such “dynamic intra-molecular conformational change” to “dynamic intrer-molecular interaction change” between a set of two specific electron transfer proteins (So far, azurin - cytochrome c).
Figure 1. In natural sensor proteins, a conformational change in the protein induced by an initial sensing event serves as a switch to turn on /off function of secondary signal generator.
Figure 2. Our approach is to reproduce the intra-molecular conformational change in sensor proteins as an inter-molecular interaction change modulable by a signal responsive molecule.
Figure 3. A signal transduction system consisting of Azurin-cytochrome c and PNIPAM. (a) PNIPAM shows temperature dependent morphological change acroos (32°C); the lower temperature: elongated random coil, the higher temperature: aggregate form (b) At the higher temperature that 32°C, the electron transfer rate dropped by 3/4.
The dynamic interaction change is realized by an environment response molecule introduced to the interaction of one of the protein (Figure 2). A change in the Inter-protein interaction induces a change in the electron transfer rate change, which can be converted to an electrochemical signal when integrating the system on an electrode. ewe varied protein. As this mechanism is simple, we believe that this is applicable to a variety of environmental responsive molecules that shows morphological change upon sensing a specific substance. In the previous research, We has succeeded in building a system using the temperature responsive polymer, poly-isopropyl acrylamide (PNIPAM), where an electron transfer rate between two proteins is clearly altered depending on the temperature (32°C) at which the morphology of the PNIPAM transforms (Figure 3). Our current approach is to build-up the mechanism which can measure the change in the electron transfer rate dependent temperature on an electrode. Thereby, the change of electron transfer rate can be detectable as a change in the electric current. In addition to the environment responsive molecule, such as PNIPAM, application of a peptide and oligo-DNA, having specific affinity to a target protein is in progress. Through such challenges, we verify the versatility of our system that can readily convert a variety of environmental signals to electrical signal.
- L. Murphy et al., Biosensors and bioelectrochemistry, Curr. Opin. Chem. Biol. 2006, 10, 177.
- Benson, D. E., Conrad, D. W., de Lorimier, R. M., Trammell, S. A., Hellinga, H.W., Science 2001, 293, 1641.
Our paper concerned with this study）
- Azurin-Poly(N-isopropylacrylamide) Conjugates by Site-Directed Mutagenesis and their Temperature Dependent Behavior in Electron Transfer Processes Angew. Chem. Int. Ed. 2009, 48, 1946-1949, Rosenberger N., Studer A., Takatani N., Nakajima H., Watanabe Y.
Searching for “intelligence: Azurin–PNIPAM conjugates were prepared by site-directed mutagenesis followed by protein reconstitution by using imidazole-conjugated poly(N-isopropylacrylamides). The polymer-bound imidazole acts as a ligand in the active site of the blue copper protein azurin. The bioconjugates showed thermosensitive behavior in electron-transfer processes with reduced cytochrome