Structure determination of GFP-like proteins

Green Fluorescent Protein-like proteins have been discovered and developed to become indispensable markers for visualizing tools in life sciences. On one hand, scientists have engineered and searched novel fluorescent proteins with diverse properties. On the other hand, the optical properties, as well as the mechanism for the photochromic behavior of fluorescent proteins have been studied comprehensively. In both approaches, the structural information mostly obtained from X-ray crystallography is essential and provides scientists the exact view on the interesting nature of these proteins. Chapter 1 presents an overview of the structural basis of the GFP-like proteins. In the following chapter, the fundamentals of crystallography in general and of the macromolecular crystallography in particular are introduced. Chapter 3 describes two structures of yellow fluorescent proteins, named SHardonnay and SHardonel, they are both the single mutants of enhanced Yellow Fluorescent Protein (EYFP), EYFP-Y203F, but showing a different first hyperpolarizability. EYFP is one of the brightest yellow FPs, however, it displays a weak second-harmonic signal by having an inversion center in its chromophore. This point mutation Tyr203Phe, aiming to eliminate the inversion center, is demonstrated in both structures. The 1.95 Å and 2.35 Å structures of SHardonnay and SHardonel, respectively, show the conservation of the yellow emission chromophore, but with a small displacement, resulting in the elimination of the inversion center. Both structures show the structural evidences for the improvement of the second harmonic signal of these variants, which make these proteins promising for second-harmonic imaging microscopy. Interestingly, the structure of SHardonel shows more than one type of oligomerization in the crystalline state, which could be responsible for the difference of the first hyperpolarizability between SHardonnay and SHardonel. The next chapter concentrates on Dronpa, the most effective reversibly switchable fluorescent protein up to date. By solving the structure of Dronpa, followed by several structures of Dronpa mutants, the structural basis of the reversible photoswitching of Dronpa has been revealed. Most of the mutations are placed in the proximate environment of the chromophore of Dronpa, with a pronounced impact on the photoswitching rate of the protein. However, PDM1-4 is a point mutant (K145N) of Dronpa in a position not involved in the microenvironment of the chromophore, but it still has an impact on the photoswitching rate of the protein. In the context of this thesis, the structure of PDM1-4 is solved to address the role of the single mutation Lys145Asn on the photoswitching rate of the protein. It is shown in the structure that the mutation does indeed not influence the conformation of the chromophore and its microenvironment, but it influences the tendency of oligomerization of PDM1-4 compared to Dronpa. The oligomerization consequently has an impact on the switching dynamics of the protein by preventing the possibility of the b-barrel to become flexible in the off-state of PDM1-4, as well as in Dronpa. Finally, chapter 5 focuses on another mutant of Dronpa, created to introduce a photoconversion into its intrinsic photoswitchable property, dubbed pcDronpa. The key mutation C62H is to modify the chromophore, followed by four other substitutions V60A, N94S, N102I, and E218G. Spectral characterization shows that pcDronpa can convert from the green-emission state to the red-emission state upon illumination with UV light, and switch from a green-fluorescent emission state to a non-fluorescent emission state like its precursor Dronpa. The structures of three states of pcDronpa (green-on, green-off, and red-on states) have been solved and analyzed in this last chapter. The modified chromophore, cyclized from the triad His62-Tyr63-Gly64, responsible for the photoconversion of pcDronpa, is well defined in the 1.95 Å structure of green-on pcDronpa. The structure shows the spatial influence between the new chromophore histidine moiety and its proximate environment, especially residues Met40 and Tyr116. Additionally, the red-on chromophore is well established in the 2.05 Å structure electron density map of red-on pcDronpa, and shows the cleavage of the backbone. This results in the contribution of the chromophore histidine moiety to the enlargement of the p-conjugation, which is accounted for the red emission of the red-on state of pcDronpa. The 2.07 Å structure of green-off pcDronpa shows the cis-nonplanar chromophore, which is the result of the cis-trans isomerization of the chromophore. This is accompanied by the rearrangement of the chromophore's microenvironment to adapt the new position of the green-off chromophore. These results on one hand demonstrate the structural basis for the photoconversion and photoswitching of pcDronpa, and on the other hand provide a useful guidance for designing better probes for advanced imaging techniques in the future.

Publication year:2013

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