Dimensión Elementos tangibles

Fluorescent proteins (FPs) are circa 27 kDa proteins that absorb light of a

given wavelength (and energy) and emit light at longer wavelength (lower

energy), and, when fused to the protein of interest, have been used to

visualise the distribution of a protein of interest in cells (Deckert et al., 2006)

and tissues (Scherrer et al., 2006). The green fluorescent protein (GFP) was

al., 1962) and cloned in 1992 (Prasher et al., 1992). The wild-type avGFP had

two excitation spectra; a major and minor excitation peak at 395 nm and 475

nm, respectively (Day et al., 2009; Heim et al., 1995). The introduction of one

mutation (S65T) resulted in a shift of the major excitation peak to 490 nm and

increased brightness (Heim et al., 1995). An additional mutation (F64L)

improved the folding efficiency of GFP at 37 °C, resulting in the enhanced GFP

(eGFP) version (Day et al., 2009). The crystal structure of GFP was solved in

1996 (Ormo et al., 1996; Yang et al., 1996), which revealed an eleven-

hydroxybenzylidene)imidazolidin-5-one (HBI), which is only fluorescent if

surrounded by a properly folded protein structure (Day et al., 2009).

Mutations that changed residues around the chromophore were found to

shift the fluorescent spectrum of the chromophore, thus resulting in spectral

variants of the enhanced GFP version, including blue FP (eBFP; 383-445nm),

cyan FP (eCFP; 439-476 nm) and yellow FP (eYFP; 514-527 nm) variants (Day

et al., 2009). Additional mutagenesis and engineering studies further

improved brightness, photostability and pH sensitivity of fluorescent proteins

(Day et al., 2009; Sample et al., 2009). In addition, FPs showed tendencies to

form dimers or higher oligomeric complexes, which led to the engineering of

monomeric FPs to avoid artificial aggregation of proteins to which the FPs

were fused (Sample et al., 2009). The palette of fluorescence proteins was

increased when a fluorescent protein was isolated from the sea anemone D.

striata, DsRed, which was excited at 558 nm and emitted at 583 nm. Using a

with live cells and reduces autofluorescence interference. Extensive

mutagenesis efforts on the DsRed protein led to a series of monomeric red

FPs that emit in the orange (551-575 nm), red (576-610 nm) and far-red (611-

660) spectrum (Sample et al., 2009). Spectral variants of fluorescent proteins

allow the simultaneous labelling of two or more proteins of interest to

monitor their distribution and interaction (Falk et al., 2001; Herrick-Davis et

al., 2006; Overton et al., 2002). The fusion of a 27 kDa protein to a GPCR

protein results in a large fusion protein that is desired to behave in a similar

manner to its untagged counterpart. Indeed, comparable ligand binding

GFP 1 2-adrenoceptors and 1 2-adrenoceptors that were not tagged with GFP (McLean et al., 2000).

H GFP

has been observed to be slower than untagged receptors (McLean et al.,

2000). This may not be unexpected, as the C-terminus of a GPCR plays an

important role in initiating internalisation and degradation processes.

An alternative strategy to fluorescently label a receptor uses the SNAP-tag

that is fused to the N-terminal end of a protein of interest. The SNAP-tag

technology is based on the human DNA repair protein O6-alkylguanine-

transferase (hAGT) which removes an alkyl group from a guanine base of DNA

and transfers it onto a reactive cysteine residue within itself (Figure 1.4) in a

covalent thioether bond (Pegg, 2011). Human AGT (207 amino acids) can also

react with O6-benzylguanine (BG) substrates (Pegg, 2011). Keppler et al. (2003)

labelling of hAGT fusion proteins in vivo (Keppler et al., 2003). Subsequent

protein engineering led to the smaller 20kDa (180 amino acids) SNAP-tag and

increased enzyme activity compared to wild-type hAGT (Juillerat et al., 2003;

Juillerat et al., 2005; Keppler et al., 2003), thus enhancing specificity of

protein labelling. The observation that the nature of the label does not

influence the reaction rate of hAGT with its substrate led to the synthesis of a

great variety of SNAP-tag fluorescent labels (excitation range from 360-782

nm commercially available), which makes this technology particularly flexible

and adaptable to experimental needs. Another main advantage is the use of

cell impermeable substrates to label SNAP-tagged membrane proteins. The

SNAP-tag is chemically inert to other biomolecules such as double-stranded

DNA and, it is not restricted to a certain subcellular location (Juillerat et al.,

2005). The irreversible and specific labelling of target proteins, which are

preferentially N-terminally fused to the SNAP-tag (Tirat et al., 2006), has been

successfully used, for example, to create an EGFR-specific imaging probe by

fusing the SNAP-tag to a single-chain antibody fragment (Kampmeier et al.,

2010). The applications of the SNAP-tag have been broadened with the

engineering of the CLIP-tag, which is another hAGT mutant. Gautier et al.

(2008) introduced eight mutations to redesign the active site of the SNAP-tag,

resulting in specificity for benzylcytosine (BC) derivatives (Gautier et al., 2008).

Using the CLIP-tag in conjunction with the SNAP-tag allows the simultaneous

labelling of target proteins with two different fluorophores for co-localisation

Alternative peptide- and protein-tags include His-tag (6 amino acids, oligo-

histidine sequence that binds to nickel or cobalt and uses nickel-

nitrilotriacetic acid fluorophores such as NTA-FITC-Ni2+) and HaloTag (297

amino acids, mutated haloalkane dehalogenase that covalently binds

fluorescent haloalkane ligands) to label proteins of interest (Bohme et al.,

2009; Los et al., 2008). Fluorescently labelled antibodies may also be used

that either directly or indirectly (i.e. one or two antibody method) recognise a

specific epitope which may be a part of the receptor of interest or an

engineered tag such as HA-tag, FLAG-tag or c-myc-tag at the N-terminus of

the receptor (Bohme et al., 2009). Most fluorescent tags require the fusion of

the tag to the protein of interest, which involves generation of a DNA

construct and its transfection into a host cell line, thus this method does not

have the potential of labelling native receptor in native cells, but is invaluable

in establishing an understanding of fundamental properties of cellular and

Figure 1.4 Schematic representation of the SNAP-tag labelling reaction. The benzylguanine (BG) SNAP-tag substrate is linked to a fluorophore. In a suicide reaction by the SNAP-tag, the fluorophore is covalently linked to the SNAP-tag, thus labelling the receptor. The reaction releases a free guanine molecule. Taken from Tirat et al. (2006).