GFP protein

Figure 11.5 Positive ion DIESMS spectra of the crude cell extract of an E. coli strain that expresses the GFP protein, showing protein peaks. The inset is the MaxEnt decon-voluted spectrum that shows more than three peaks attributable to proteins from the extract.

In the years immediately following, this procedure was optimized to express real proteins, the first being - for obvious detection reasons - the green fluorescence protein (GFP). Protein expression is actually the subject of the next section. 

Green fluorescent protein is commonly used for energy-transfer experiments (Baubet etal. 2000). The fluorescent moiety of GFP protein is the Ser-Tyr-Gly derived chromophore. GFP can be expressed in a variety of cells where it becomes fluorescent, can be fused to a host... 

In comparison to equivalent optical detection methods using whole cell biosensors for water toxicity detection, these results proved to be more sensitive and produce faster response time. Concentrations as low as 1% of ethanol and 1.6 ppm of phenol could be detected in less than 10 min of exposure to the toxic chemical, whilst a recent study [11] which utilized bioluminescent E.coli sensor cells, detected 0.4 M (2.35%) ethanol after 220 min. An additional study [1] based on fluorescent reporter system (GFP), enabled detection of 6% ethanol and 295 ppm phenol after more than one hour. Cha et al [12] used optical detection methods of fluorescent GFP proteins, detected 1 g of phenol per liter (1,000 ppm) and 2% ethanol after 6 hours. Other studies [13] could not be directly compared due to different material used however their time scale for chemicals identification is hours. 

Ramachandran-type plots in which the t and (p dihedrals of the chromphore within the GFP protein matrix were systematically varied [50] showed that there are two minima for all protonation states, one at z = 60 30° and

protein environment of GFP allows the chromophore some rotational freedom, especially by a HT or in the (p dihedral angle (Fig. 5.7). There is a significant energy barrier for t = 180-270°, therefore a cis-trans photoisomerization cannot occur by a 180° rotation of the (p dihedral angle. The protein exerts some strain on the chromophore when it is planar, and the only reason planar chromophores are found in GFP is due to their delocalized -electrons. These results have been confirmed by molecular dynamics simulations of the chromophore with freely rotating t and cp dihedral... 

Transfection and expression of the GFP protein decreased exponentially in irradiated samples, but at a slower rate than the loss of supercoiled forms. This resulted in a target size considerably smaller than the size of the DNA molecule, only about one-third the mass. The map of this plasmid (Fig. 4) shows that the segment of the plasmid directly involved in transfection activity (which includes the CMV promoter, GFP gene, SV40 intron and polyadenylation signal) has a calculated mass of 1067.880 kDa. 

The case of the GFP chromophore is important since its gas-phase spectra are available and one can make a direct comparison with the experiment at various level (gas phase, solution, protein matrix) [27,49]. Such comparison is schematically reported in Scheme 12.4. Inspection of these data reveals that the gas-phase absorption maximum is substantially closer to the protein absorption maximum than to the solution absorption maximum. This suggests the rather naive idea that the GFP protein cavity offers an environment more similar to the gas phase than to the solution. 

However, it rapidly turned out that expression of wildtype GFP often results in poor fluorescence yields and that the system is rather insensitive. Further investigations proved that thermosensitivity of GFP protein maturation is one of the major problems leading to the accumulation of improperly folded non-fluorescent, insoluble protein. [13] Several research groups addressed this problem and tried to identify GFP mutants possessing improved properties such as improved light emission properties (higher quantum yield and/or a extinction coefficient) and/or solubility, which both should increase the amount of detectable fluorescence considerably. In. addition, research also focused on the identification of GFP mutants with altered spectral properties (e.g. altered emission peak wavelength) in order to create... 

As yet, for the majority of other GFP proteins temperature stability has not been investigated. 

Scheme 10 Semisynthesis of lipidated proteins by using EPL (a) geranylgeranylated Rab7, (b) famesylated Rheb, (c) famesylated K-Ras4B, (d) (e) PE-modified GFP protein, (f)...

Scheme 15 Site-specific lipid attachment through sortase-mediated transpeptidation. (a) Mechanism of sortase-mediated ligation, (b) Semisynthesis of lipid modified GFP protein by sortase-mediated ligation, (c) Semisynthesis of lipidated K-Ras4B protein, (d) Semisynthesis of GPI modified GFP protein...

Fig. 5 Structures of native GPI-anchor (86) and GPI-anchor analogues (87), (88), and (89). These structures contain three domains of GDI-anchor (1) a phosphoethanolamine linker (red), (2) the common glycan core (black) and (3) a phospholipid tail (blue). R is a GPI anchor side chain, such as galactose or phosphoethanolamine. The GPI-analogues were attached to GFP protein by EPL to produce GFP-2 (87), GFP-3 (88), GFP-4 (89)...

Hnally, we note that GFP can be used as a tag to allow easy Election of gene expression. In diese cases die desired graes are linked to die gene for GFP. Protein syndie-sis can be readily monitored from die amount of GFP formed in the incubator Fhioresoence sensing is... 

Based on the Amaryllidaceae alkaloid galanthamine, a biomimetic solid-phase synthesis of 2527 compounds was reported by Shair and coworkers (Figure 11.13) The core scaffold, initially prepared in several steps, was diversified by means of four successive reactions Mitsunobu reaction of the phenolic moiety with five primary alcohols, Michael addition of the a, 3-unsatnrated cyclohexenone with thiols, iV-acylation or A -alkylation of the cyclic secondary amine, and treatment of the ketone with hydrazines and hydroxylamines. Further evaluation of library constituents for their ability to block protein trafficking in the secretory pathway of mammalian cells led to the discovery of sercramine as a potent inhibitor of the VSVG-GFP protein movement from the Golgi apparatus to the plasma m brane. 

Figure 12.15 AFM images of (a) a GFP protein array deposited with a 220 nm aperture NADIS tip (spot diameters 150 nm) (b) the same with a 110 nm aperture NADIS tip (spot diameters 30 nm) (c) zoom on a 25 nm spot and height profile (d) NP array obtained with a 300 nm aperture NADIS tip (e) the same with a 130 nm aperture tip and (f) zoom on spots with one and two NPs.

In order to examine the surface modifications of other thiol-organosilica nanoparticles with proteins, the nanoparticle solutions were mixed with GFP/protein solutions and evaluated with fluorescence microscopy. The thiol-organosihca nanoparticles were shown to be well dispersed, with a distinct fluorescence (Figure 4.9), which indicated an effective modification with GFP and retention of dispersion this contrasted with the TEOS nanoparticles, which showed no clear fluorescence (Figure 4.9). 

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