The biology of light

December 16, 2011

If you want to know something about the connection of biomolecules and light, then this might be of interest to you. I also thought it is pretty interesting, so I wrote this piece on proteins, linker-molecules, emission, and fluorophores. First of all because I wanted to understand the principles of fluorescence better since it is important for my project (FRET and confocal microscopy), but secondly also because I like the interplay between biology and physics. For my work on the hypothetical interaction of Staphylococcus areus SecA1 and SecA2 proteins I use two molecules that have fluorophore properties. Fig.1 depicts these molecules called fluorescein-5-maleimide (A) and Cy5-maleimide (B). Obviously both names are not the IUPAC nomenclature names for chemical compounds, however in everyday lab-life, these two names seemed more practical.

 

Fig. 1: Chemical structure of two fluorophores that can be used to label proteins via sulfur containing thiol groups on external cysteines. Left fluorescein-malamide is depicted and on the right side Cyanine (Cy5) maleimide. The maleimide group is the lowest identical portion of both molecules.  It functions as a linker between the thiol group and the actual fluorophores.

To be fluorescent actually describes a property that some molecules have. This includes the absorption of light at a specific wavelength (relatively high energy) and the subsequent emission of light with a longer wavelength and lower energy per photon. The relation between the energy of a photon and its wavelength is important when talking about fluorophores (Fig. 2). The frequency of a photon (≈ internal energy) is inversely proportional to its wavelength, thus photons with a short wavelength contain more energy than photons with longer wavelength.

 Fig. 2: The relation of a photon energy with its wavelength. Long wavelength means lower energy, because the frequency of the photon decreases (not shown in this formula) (1).

If one concentrates on the chemical structures depicted in Fig. 1 it becomes apparent that they both contain a large number of double bond containing cyclic carbon rings. And this is also the case for most fluorophores in nature. These structures are one secret of fluorescent properties.

Light low wavelength transfers energy on electrons in double bond which becomes excited and “jumps up” a band, thereby stabilizing the energy which has just been absorbed. This excited state of the electron does not last forever and it will fall back to its original and more stable state sooner or later. However, thermodynamics tells us that energy does not just disappear so the energy which is lost (because the electron has fallen back) must be somewhere. Indeed the energy is somewhere. The fluorescence effect that we see is the electromagnetic radiation in the form of photons that is emitted from the falling electron. Important to notice is, however, that not all energy is emitted as visible electromagnetic radiation. The physical turnover processes also create other emissions that cannot simply be seen by eye and in addition energy is “lost” through collisions of our fluorophores with other molecules. Since this is the case, the emitted energy is somewhat lower than the absorbed energy and therefore also the wavelength of the emitted photons becomes longer, as Fig. 2 tells us. As light color depends on wavelength, the emitted color that can be observed is different from the previously absorbed one (Fig. 3).

 Fig. 3: Emission spectra of green fluorescent protein (GFP). The wavelength of the emitted light is longer than the wavelength of the absorbed light. Therefore also the color of the light appears different (2).

Fluorescent properties of molecules enable biologists to mark proteins or other biomolecules in order to make them easily detectable by eye or with the help of photomultipliers. Two or more fluorescent markers can even be combined and might transfer energy to each other when their carrier molecules make contact. Changes emission spectra can consequently be observed. This is the basis of FRET microscopy and allows me to study the potential dimerization of proteins that are thought to function in the same protein translocation pathway.

(1)http://astronomyonline.org/Science/Frequency.asp

(2)http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/Product-Spectra.EGFPpH7.html

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