Resultados por Dimensiones de las Variables Motivación y Liderazgo Transformacional.

Fluorescence occurs when the absorption of light causes a molecule’s electronic configuration to enter an excited state and the spontaneous return to the ground state results in the emission of a photon of lower energy (higher wavelength) than the incident photon [173]. The ability to label proteins with fluorescent dyes or naturally-fluorescent proteins has allowed scientists to track changes in location of proteins in real time, to measure the subcellular compartments where proteins are activated in vivo, and to monitor protein-protein interactions in real time [174, 175]. Generally when a dye is excited with a excitation beam from a non-laser excitation source, the dye is excited by a random orientation of incident photons. It was noted in 1926 by Francis Perrin that, if an immobilized fluorophore is excited with plane-polarized light, the emission is polarized in the same plane; however, if the fluorophore is in solution, the emission becomes random [176]. This phenomenon has been extremely useful in the biological sciences to track single nucleotide polymorphisms, peptide/protein interactions, DNA/protein interactions, phosphorylation events and photolytic cleavages [177-181]. Two different, but interchangeable, methods are commonly used to quantify this phenomenon: anisotropy (r) or polarization (P). In general, biophysicists prefer to use anisotropy because the loss of light due to the polarizer is corrected by the factor of 2 in the denominator of the equation, while polarization (P) has become the standard in the biological sciences [182]. Both anisotropy and polarization measurements are made by

quantifying the intensity of the fluorescence emission perpendicular (I_&) and parallel (I||) to

the plane of excitation (Equation 1.1; Figure 1.11); the value of anisotropy and polarization can be easily interconverted (Equation 1.2). The remainder of the text will refer to polarization in terms of (P) which, while technically unitless, is commonly expressed in the literature as milliP (mP). (1.1) ! r= I||"I# I||+2I# ! P= I||"I# I||+I# (1.2) ! r= 2 P

( )

Given that excitation, relaxation, and subsequent emission do not occur instantaneously, if a fluorescent dye is undergoing rotational motion, a polarized excitation source will be depolarized if the rotational motion of the dye is faster than its fluorescence lifetime. Polarization measurements provide an index of the average angular displacement of a fluorophore that occurs between absorption and emission of a photon. The angular displacement and, thus, the polarization is dependent on the rotational velocity of the molecule (rotational correlation time; '), the delay of the fluorophore from excitation to emission (fluorescence lifetime; (), and the fundamental polarization for a particular dye (physical constant for a particular dye; P0), as set forth by the classic Perrin equation (Equation 1.3) [176, 182]. (1.3) ! 1 P" 1 3 # $ % & ' ( = 1 P₀ " 1 3 # $ % & ' (

(

)

The rotational correlation time (') is dependent on the viscosity (!) of the environment and the apparent molecular weight of the fluorescent dye or dye-conjugate (M). For a globular

protein, the rotational correlation time (") is directly related to the molecular weight of the protein by the formula:

(1.4)

!

" =#M

RT (v +h)

Typically in the biological sciences, fluorescence polarization is used for measuring binding reactions in which viscosity of the solution remains unchanged. Therefore, the only variable that is commonly changing the rotational correlation time (") is the apparent molecular weight of the dye. The most widely used dye for fluorescence polarization is fluorescein isothicyanate (FITC) and its derivatives which have a fluorescence lifetime (() of ~4 ns. This fundamental property of the dye limits the types of molecular interactions that can be monitored using fluorescence polarization (Figure 1.12) [182]. For instance, a FITC-labelled molecular probe in general must have a molecular weight below 5,000 Da and bind to an interactor of greater than 10,000 Da in order to obtain a sufficient signal by fluorescence polarization (Figure 1.12). While there are no commercial fluorophores available with fluorescence lifetimes that allow the detection of interactions between two large macromolecules, several publications exist describing novel dyes capable of monitoring binding interactions between albumin and antibodies [183-185].

While fluorescence polarization assays require that the molecular weight of the probe must be much less than the molecular weight of the bound complex, this limitation is offset by several advantages that fluorescence polarization offers compared to traditional methods for monitoring protein/ligand and protein/protein interactions. First, no radioactive waste is generated while the probe concentration can remain low, typically in the picomolar to nanomolar range. Second, fluorescence polarization assays are homogeneous and do not require additional steps after the reaction is established for separating the tracer from the

reaction mixture. In addition to equilibrium binding analyses, fluorescence polarization allows the experimenter to set up experiments so that kinetic data can be obtained from multiple samples (including 96 well, 384 well, and 1536 well formats). Given that the polarization signal is not dependent on the absolute intensity of the fluorophore, these fluorescence polarization assays are able to accommodate day-to-day variations in probe concentration, as well as loss of probe due to decay, and are generally robust to instrument changes such as drift, gain settings, and lamp changes [186]. In Chapters 4 and 5, I describe my use of fluorescence polarization in developing two different assays, namely the binding of G!i1(GDP) to the GoLoco motif of RGS12, and the production of GDP by RGS4-