Research topics of Nakajima

Biosensors that use enzymatic reactions are capable of highly selective substance detection because of the inherent substrate recognition ability (Ref. 1). A sensor generally consists of a sensing part that detects the presence or absence of a substance or a change in the environment and generates some primary signal. Then,  a signal conversion part converts the primary signal into a measurable secondary signal. Electrical signals, readily detectable and processable, are widely used as secondary signals. This point is also true of biosensors exploiting enzymes. However, since the enzymes do not have a signal conversion system as their original function, the primary signal generated at the sensing site (the enzyme reaction) should also be the second signal. In this context, most of the enzymes used for the sensor are accompanied by redox reactions, limiting the application of biosensors.

 Meanwhile, sensor proteins in nature share the general operating mechanism: generation of a primary signal (some reaction such as bond formation and/or cleavage) on a sensing event >> some change in protein structure >> generation of secondary signals. In these sequential processes, the structural transformation of the protein corresponds signal transmission and conversion. Many studies on the sensor proteins have revealed that the signal transduction process; the higher-order structural change originates from a local structural change that occurs at the sensor site as some chemical reaction (Fig. 1). Artificial reproduction of such high-order structural changes may allow the creation of novel biosensors with a signal transduction mechanism and expand applications of biosensors. However, it is not easy to artificially design the mechanism equivalent to that of nature at the molecular level, and related studies are hardly known so far (Ref. 2). To settle this problem, we devised to construct the signal conversion process of the sensor proteins with "dynamic change in molecular interaction between specific proteins." Concretely, we build a dynamic interaction system consisting of a set of electron transfer proteins (currently azurin and cytochrome c) and an environment-responsive molecule (Fig. 2, Ref. 3). In this system, tentative and specific interaction that electron transfer proteins form in the electron transfer processes is used as a 'higher-order structure.' This interaction is linked to the sensing process of the environmentally responsive molecule introduced into one of the proteins. Since some change in the protein-protein interaction should affect the electron transfer rate between the proteins, the sensing process by the environment-responsive molecule is converted into a measurable electrical signal. In this system, the inter-protein interaction change is a key to the signal conversion, and we believe that this architecture is applicable to various environment-responsive molecules that show some morphological changes upon the sensing process. Our previous studies have already succeeded in constructing a system using a poly isopropyl acrylamide (PNIPAM) analog, a heat-responsive polymer, as an environmental response site. We confirmed a clear change in the electron transfer rate upon the temperature change crossing the morphological change point (34ºC) (Fig. 3). In a recent study, we have tried integrating this signal transduction system on an electrode so that the alteration in temperature can be monitored as some change in the electrode current.

 In addition to environmentally responsive molecules such as PNIPAM, oligo-DNA and peptides are used as environment responsive sites. The presence of the molecules and proteins that specifically bind to these sites is detected by a change in electron transfer rate and electric current(Ref. 4). Expanding the application fields of biosensors is a popular topic of chemistry, while the main achievements are to discover new enzymes applicable to the biosensors and the electrode materials for detecting enzyme reaction products. Most of these studies presuppose the mechanism of existing biosensors: the signal conversion mechanism does not exist in enzymes. Our approach is inspired by the signal conversion mechanism of natural sensor proteins. Currently, optical methods such as changes in fluorescence are in the mainstream for constructing DNA and peptide chips. The developed mechanism may provide a method to detect the biological polymers as electrical signals directly.

References)

1. L. Murphy et al., Biosensors and bioelectrochemistry, Curr. Opin. Chem. Biol. 2006, 10, 177.
2. Benson, D.E., Conrad, D.W., de Lorimier, as a few examples. R. M., Trammell, S. A., Hellinga, H.W., Science 2001, 293, 1641.
3. Rosenberger N., Studer A., ​​Takatani N., Nakajima H., Watanabe Y., ACIE 2009, 48, 1946. Nakajima H., Miyazaki S., Itoh T., Hayamura M . Watanabe Y., Chem. Lett. 2014, 43, 1204,

In general, proteins are considered to be fragile and readily lose their original functions under conditions other than a neutral aqueous solution and near room temperature. This concept originates from our daily experiences that the proteins we usually see are derived from cells that live in the same growth environment as our cells. Meanwhile, proteins produced in extremophiles, which grow in harsh environments from our viewpoint, exhibit extreme stability and durability of functions under certain conditions. Thermophiles are a useful example of extremophiles (Ref. 1). Thermus thermophilus HB8, a thermophile used in our study, was originally found in a fumarole of hot springs in the Izu district. This bacterium is thermally tolerant even in boiling water. Many thermophile-derived proteins have excellent durability and organic solvent resistance, overturning our idea that "proteins are fragile and unusable" and allowing the proteins as chemical materials in various fields. Thus, a goal for chemists is to fabricate novel functional proteins while maintaining and utilizing the excellent properties of the thermophile. The following are advantageous properties of using thermophile-derived proteins as the platform for artificially functional proteins.

1. In many cases, a large amount of protein can be obtained as a recombinant using E. coli as a host strain.
2. Since the protein is stable even at high temperatures, it is relatively easy to purify and handle after purification processes.
3. The proteins exhibit tolerance towards multiple mutageneses while maintaining the original higher-order structure and functions, allowing the facile molecular design to impart artificial functions to the proteins.

 Although 1) and 2) do not seem to be the essence of research, the ease of production and purification is important in daily research works. The following is a brief introduction to our research using thermophile-derived proteins. From the thermophilus-derived proteins, we selected cytochrome c₅₅₂ (Cyt c₅₅₂), an electron transfer protein whose three-dimensional structure has been well-determined. This protein has heme, an iron protoporphyrin derivative, as a cofactor. Our first study with this protein was to create a heat-resistant peroxidase. The peroxidase contains a general acid-base catalytic mechanism as the key in the catalytic cycle. As the electron transfer protein Cyt c₅₅₂ does not have such a mechanism, we decided to introduce the catalytic mechanism in the heme vicinity by mutagenesis (Ref 2-5). Taking a closer look at the three-dimensional structure of the protein, Val49, located approximately 5.2 Å above heme iron, was replaced with aspartic acid. The heme of Cyt c₅₅₂ has an octahedral hexa-coordinated structure, and there is no coordination site on the iron ion for a chemical reaction. Therefore, we replaced the original Met69 with alanine and made a vacant site on the iron ion. The peroxidase activity of this mutant is shown in Figure 1. The catalytic activity of the V49D/M69A mutant (Fig. 1a, ○) increases with increasing temperature, and the increase in reaction rate due to heat is reflected in the catalytic activity. Compared to previously reported mutants of myoglobin H64D, which shows high peroxidase activity comparable to natural peroxidases (Figure 1a, △), Mb H64D outperforms catalytic activity up to 40ºC, while the V49D/M69A mutant shows the superior activity to the myoglobin mutant at 60 and 70ºC. The sharp decrease in the activity at 80ºC is due to the formation of a 6-coordinated heme species by the unidentified ligand. This blocks the active site without the protein denaturation. Therefore, lowering temperature revives the catalytic activity. The superior property of the V49D/M69A mutant observed in the catalytic rate at high temperature is also found in the persistence of the activity. Figure 1b shows the time course of substrate consumption monitored during the peroxidase reaction at 70 ºC using the V49D /M69A and Mb H64D mutants as the catalysts. The V49D / M69A mutant always outperforms the myoglobin mutant in substrate consumption. The result indicates that the V49D/M69A mutant has durability in the catalytic activity under high-temperature conditions. The initial heat resistance of the protein is also effective for catalytic activity in high temperatures. Other research projects using the Cyt c552 are also in progress (Ref. 6).

References)

1. Nakajima, H., Shoji, O., Watanabe, Y., Catal. Surv. Asia 2011, 15, 134. 
2. Nakajima, H., Ramanathan, R., Kawaba, N., Watanabe, Y., J. Chem. Soc. Dalton Trans. 2010, 39, 3105. 
3. Nakajima H., Ichikawa Y., Satake Y., Takatani N., Manna SK., Rajbongshi J., Mazumdar S., Watanabe Y., ChemBioChem 2008, 9, 2954. 
4. Watanabe Y., Nakajima H. and Ueno T., Acc. Chem. Res. 2007, 40, 554. 
5. Ichikawa Y., Nakajima H., Watanabe Y., ChemBioChem 2006, 7, 1582. 
6. Ibrahim, Sk. Md., Nakajima, H., Ramanathan, K., Takatani, N., Ohta, T., Naruta, Y., Watanabe, Y., Biochemistry 2011, 50, 9826.