Mysteries in Science: Concerted Evolutionary Engineering of Proteins and Small Molecules

Takeaki Ozawa

(Professor, Department of Chemistry)

Chemistry helps us to understand natural phenomena in terms of their constituent atoms and molecules, and its greatest gift is that it allows us to create completely new substances that are useful to humanity. For example, we can artificially synthesize natural polyether compounds with molecular weights in the thousands, and some of these techniques have found application in drug development. However, life creates even larger proteins with molecular weights of 10,000 or more which have advanced functions that cannot be replicated by artificial means. How such protein functions were created from atoms in the process of evolution remains a mystery.

Among this diverse range of functional proteins, we focus on the photoreceptor proteins that absorb or emit light, and seek to use these proteins in analytical techniques in life science research. As an example, let’s take the source of firefly light. Fireflies emit light by converting the energy produced in a chemical reaction between the luciferase and its substrate luciferin, into light. In the distant future, we may be able to artificially create a luciferase-like enzyme with the desired luminescence properties from scratch. However, it is also possible to artificially evolve luciferase — created naturally in the process of evolution — to give it new functions. One of the simplest examples of this involves changing the wavelength of the light emitted by luciferase by introducing random mutations into the luciferase gene. Another technique involves creating a cyclic luciferase molecule in which its ends –the amino terminus and carboxy terminus – are joined together by a protein splicing reaction with intein. Cyclic luciferase does not emit light because of structural hindrance, but when it is cleaved by an enzyme such as a protease, its activity is restored and light is emitted. In another example, luciferase loses its activity when it is split into two fragments at a specific location, but recovers that activity when the split ends come close and the fragments are reconstituted into the native form. This splitting and reconstitution of luciferase has been used to detect protein interactions and post-translational modifications. Fireflies have the property of flashing their light on and off, and it is also possible to artificially create a flickering phenomenon by inserting a plant-derived photosensitive protein at a specific site in luciferase. This modified luciferase has been used as a sensor for measuring pH levels in living organisms. Thus, the artificial evolution of luciferase has led to the development of technologies to detect various phenomena in the living body as luminescence signals [1].

Proteins are not our only targets for evolution. Researchers have also attempted to control the color of light emitted by luciferase by artificially modifying its substrate. One drawback, however, has been the fact that the modified substrate is less adjusted to luciferase, and hence its activity is greatly weakened. We are therefore using evolutionary engineering techniques to improve luminescence by evolving luciferase to suit the modified substrate.

Such evolutionary engineering of photosensitive proteins has been achieved not only for luciferase but also for various fluorescent proteins and plant-derived photoreceptor proteins. The latter have attracted a great deal of attention in the field of optogenetics, and will become even more important in the future [3]. The evolutionary engineering of both proteins and binding molecules may create proteins with functions that go far beyond the limits of our imagination. To achieve this goal, technological innovations that further accelerate molecular evolution will be key.

References

1. M. Endo and T. Ozawa, “Strategies for development of optogenetic systems and their applications.” J. Photochem. Photobiol. C, 30, 10-23 (2017).

Figure. Evolutionary engineering of luciferin and luciferase.

― This article is from the "Mysteries in Science" series in The Rigakubu News ―

Translated by the Office of Communication

― Office of Communication ―