GIMP my protein

September 8, 2012

While playing around with the open-source GNU Image Manipulation Program (GIMP) I asked myself: Why not transform proteins into a construct that does not resemble the standard Protein Data Base structures? Well and this is the humble result: Another perspective on the famous green fluorescent protein, which has become so important for many projects. The GFP molecule you are seeing above is actually a modified version of the “real” protein. By modifying certain amino acids researchers around Jenny J. Yang (Georgia State University, Department of Chemistry) managed to lend calcium and proteinase detecting properties to GFP. GFPs can then be incorporated into tissues were it is important to monitor calcium concentrations. In many organisms calcium is used to build up gradients to for example transmit chemical “information” as in nerve cells. Therefore, this type of protein, next to its funny looking barrel shape, also offers some handy features to work with and might help to detect diseases such as cardiomyopathy Alzheimer’s on a molecular scale (= earlier).

With the fitting amino acid mutations GFP can not only be used for the detection of calcium ions, but also for the detection of other ions. A very famous ion is the hydrogen ion. The concentration of hydrogens ions determines the pH (yes, that’s the H in pH). The following few lines might be a bit technical, but they explain why GFP is loved by so many scientists. And why it is a perfect example of a biomolecular structure-function relationship. High pH sensitivity and specificity, rapid signal response and good optical properties are important characteristics for a pH sensor. In addition it must be possible to reach  intracellular sites in a non-invasive and non toxic manner. GFP fulfills many of these prerequisites and therefore has been used as intracellular pH sensor. But how does it work? The sensitivity and specificity of GFP mainly seems to be based on the protonation state of the phenolic group of the chromophore (the green thing in the center). In other words the charge of the light emitting parts of the GFP molecule changes. The GFP chromophore 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI) lies in the center of GFP’s β-barrel structure (made up of the white arrows) and normaly is non-fluorescent due to its unionized phenol group. However, the inward facing amino acid residues Ser65–Tyr66–Gly67 can cause the ionisation of the phenol group, leading to potential fluorescence. During different pHs, different protonation states of the chromophores phenol group are likely to influence its photochemical properties by influencing the ionisation of the phenol group. Different pH values therefore change the charges of the centrally located molecule that is responsible for the green light. A different pH value translates into a different light intensity.

Why light becomes emitted in a different manner/intensity during different protonation states is, however, a topic for a future blog entry. Only this far: The hydroxyl group of the phenol and its protonation states within the chromophore play a role, leading to different relaxation times of the electrons that become excited by the incoming laser…

Check Ulrich Haupt’s and coworkers article if you want to know more.

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