Proximity ratios

This apparent dilema was resolved when further crystallographic data on T3 compounds became available (11,12,13,14,15). (See reference 11 for a detailed list of thyroactive crystal structure studies.) These determinations show the 3 -I in the distal conformation and further showed that either the distal or proximal conformer could be isolated by changing the crystallizing media (13,14). These data prompted further NMR and MQ studies which showed a nearly equal distal/proximal ratio in solution (16) and a much smaller barrier to internal rotation (17). Thus these data have shown that the distal and proximal conformers are readily accessible in solution and that the energy barrier to free rotation is small enough so that the stereospecific conformer required by a receptor is easily accessible. 

Following the same trend as the previously mentioned catalysts, oxidation with [Fe(OTf)2( Pytacn)] occurred preferentially at the tertiary position more remote from EWGs, indicating its electrophilic nature (Scheme 27a). The remote/ proximal ratios obtained were viitually the same as those attained with catalysts [Fe (pdp)(CH3CN)2](SbF6)2 and [Fe(OTf)2((S,S,/ )-mcpp)] (for comparison see Schemes 11 and 18, respectively). However, using [Fe(OTf)2 (M N Pytacn)], a significant amount of secondary site oxidation (ketone formation) was observed. Moreover, methylene oxidation was also sensitive to the electronic properties of the C-H bonds, as shown in the oxidation of methyl hexanoate (Scheme 27b). 

Here I a and Ip are the fluorescence intensities of the donor and acceptor respectively (for the same doubly labelled molecule), 4>a and Op are the quantum yields of the donor and acceptor molecules and and rjp are the detection efficiencies of the experiment at the wavelengths of the fluorescence signals from the two molecules. For simplicity, the correction factor, y, to account for differential detection efficiencies and quantum yields of the two fluorophores is generally assumed to be unity (see for a discussion [78]), and fret is then sometimes referred to as the proximity ratio P [ 77]. 

Histograms of proximity ratios are commonly fit with single or multiple Gaussian or Lognormal curves in order to extract information on peak positions, widths and areas [76]. It is possible to describe the data with an analytic expression in some cases (see [76]), but the difference in the characteristic parameters obtained (peak position, width and area) using a Gaussian is small [ 76 ]. Care must also be taken with the construction of the histograms, with suitable numbers (and widths) of bins chosen to aid in the resolution of any components that may exist. 

The two resolved peaks have proximity ratios that are consistent with a dynamic equilibrium between the expected folded and unfolded conformations of RNA. 

The high proximity ratio species is assigned to folded molecules and the lower proximity ratio species assigned to unfolded molecules. A further discussion relating to the number of peaks that are observed, and the conditions in which one will observe distinct peaks for species in dynamic equilibrium is given in later subsections. 

In the examples described so far the proximity ratio was calculated and a threshold was applied for every bin (integration time) in the data. Such an approach has advantages and disadvantages. One must remember that the data consists of bursts above the background that, even for small molecules, can persist for several integration times (see Figure 2.1). If the diffusion rates for two... 

Figure 4.8 Surfactants prevent non-specific absorption of protein to the sampie container.The raw data (ieft, donor channei grey, acceptor channei biack) and the caicuiated proximity ratios (right, threshoid = 40 counts, see Chapter 2 for the method) both show the increase of a freeiy diffusing species with a reiativeiy high (pre-sumabiy nativeiy foided) proximity ratio upon titration of Tween 20 (0.0001 % to 0.01 %, top to bottom) into 400 pM im9S81C iabeiied with Aiexa 488 and Aiexa 594C5 maieimide, 50 mM sodium phosphate pH 7.0.

Recently, methods applied to the analysis of data from novel multiparameter experiments [7,53,54] have demonstrated a way to test for active acceptor after the measurement of the proximity ratio. This therefore allows almost complete removal of inactive acceptor molecule influenced data which therefore eliminates the zero peak altogether, although at the expense of much more complicated instrumentation. This method is discussed further in Chapter 2. 

Figure 4.9 The effect of laser power upon the photobleaching of a freely diffusing spFRET labelled protein. Upon increasing the laser power from 40 to 120 /u,W (top to bottom, laser power measured before the objective, see Chapter 3), the raw data (left, donor channel grey, acceptor channel black) and the calculated proximity ratio histograms (right) both show the increase in donor only fluorescence (increase in donor channel in the raw data, increase in the relative magnitude of the zero peak) that is indicative of photobleaching.

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