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adapted for expression in mammalian cells and characterized by a significantly red-shifted Stokes shift that emits transferred energy at 508 nm. The resonance energy transfer from the reaction of the reconstructed RLUC protein with its substrate Deep Blue Coelenterazine (DBC) excites the GFP2 protein, as the two fused proteins Id and MyoD, or FKBP12 and FRB which interact in the presence of a small molecule mediator (rapamycin). Our lab also demonstrated the ability to detect signal from PPIs in cultured cells, as well as from the surface and deeper tissues of small living animals with implanted cells over expressing the fusion constructs (For

Our lab has recently showed that the BRET2 assay sensitivity can be significantly improved by using RLUC mutants with improved quantum efficiency and/or stability (eg, RLUC8 and RLUCM) as a donor. To extend the time of light measurement, we also developed CLZ400 (also known as bisdeoxycoelenterazine) analogs, showing that signal from our improved BRET2 vector can be monitored for up to 6 hours. This approach, currently undergoing continuing validation, should have important implications for the study of PPIs in cells maintained in their natural environment, particularly if it can be effectively applied for the evaluation of new pharmaceuticals. Most recently, further advances in this field have led us to develop a high photon efficiency, self-illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant RLUC (RLUC8). This new BRET fusion protein (BRET3) exhibits a several fold improvement in light intensity in comparison to existing BRET fusion proteins. BRET3 also exhibits the most red-shifted light output (564 nm peak wavelength) of any reported bioluminescence protein that uses its natural coelenterazine substrate, a benefit that can be demonstrated at various tissue depths in small

Molecular imaging has been recognized as an important and exciting area of bio-medical research, mainly because of its ability to visually represent, characterize, and quantify biological processes in living subjects. Techniques such as Positron Emission Tomography (PET), Single-photon Emission Computed Tomography (SPECT), and Magnetic Resonance Imaging (MRI) have been extensively used in the clinic for several diagnostic and disease monitoring processes; all these systems explore intracellular proteins or other molecules as probes for the signal. Reporter genes (Bioluminescence, fluorescence, and PET), on the other hand, are capable of precisely monitoring sub-cellular processes and their native functional actions in cells, and imaging them in living animals. Challenges, however, remain in delivering these proteins in cells without perturbing the cellular microenvironments. Another obstacle is in generating sufficient sensitivity to measure theses signals, especially in living animals. Problems associated with the modulations in the cellular microenvironment are also tricky, but may be minimized by expressing few copies and weighing the sensitivity. The continuing development of new high-sensitivity instruments with tomographic imaging capabilities and improved spatial resolutions will play an important role in expanding the applications of bioluminescent reporters and exploiting their unique ability to precisely image

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**4** 

**Quantitative Assessment of Seven** 

Valentina Kubale1, Luka Drinovec2 and Milka Vrecl1

*1Institute of Anatomy, Histology & Embryology, Veterinary Faculty of University in Ljubljana,* 

*2Aerosol d.o.o., Ljubljana,* 

*Slovenia* 

**Transmembrane Receptors (7TMRs)** 

**Oligomerization by Bioluminescence** 

**Resonance Energy Transfer (BRET) Technology** 

Seven transmembrane receptors (7TMRs; also designated as G-protein coupled receptors (GPCRs)) form the largest and evolutionarily well conserved family of cell-surface receptors, with more than 800 members identified in the human genome. 7TMRs are the targets both for a plethora of endogenous ligands (e.g. peptides, glycoproteins, lipids, amino acids, nucleotides, neurotransmitters, odorants, ions, and photons) and therapeutic drugs and transduce extracellular stimuli into intracellular responses mainly via coupling to guanine

These receptors have traditionally been viewed as monomeric entities and only more recent biochemical and biophysical studies have changed this view. The idea that 7TMRs might form dimers or higher order oligomeric complexes has been formulated more than 20 years ago and since then intensively studied. In the last decade, bioluminescence resonance energy transfer (BRET) was one of the most commonly used biophysical methods to study 7TMRs oligomerization. This technique enables monitoring physical interactions between protein partners in living cells fused to donor and acceptor moieties. It relies on nonradiative transfer of energy between donor and acceptor, their intermolecular distance (10 – 100 Å) and relative orientation. Over this period the method has progressed and several versions of BRET have been developed that use different substrates and/or energy donor/acceptor couples to improve stability and specificity of the BRET signal. This chapter outlines BRET-based approaches to study 7TMRs oligomerization (e.g. BRET saturation and competition assays), control experiments needed in the interpretation i.e. establishing specificity of BRET results and mathematical models applied to quantitatively assess the

**2. Seven transmembrane receptors (7TMRs): Structure and characteristics**  Primary sequence comparisons reveal that 7TMRs share sequence and topology similarities allowing them to be classified as a super-gene family. These receptors are characterized by

nucleotide binding proteins (G-proteins) (McGraw & Liggett, 2006).

oligomerization state of studied receptors.

**1. Introduction** 

