Diffusion spFRET

Fundamental photophysical considerations in diffusion spFRET measurements... 

Abundant examples of diffusion spFRET can be found in the literature. Indeed it is in this area that single molecule fluorescence techniques have arguably proved the most useful. In Chapter 5 we review three papers in detail but here we present a very brief review of a broader range of spFRET experiments. 

One limitation of diffusion spFRET clearly seen in studies of proteins is that one requires that the states are populated for a significant time. One cannot practically study the denatured state, for example, in native buffer conditions where the population is less than a few percent. In principle, molecules in this state are measured and recorded individually but practically they would be lost in... 

Figure 3.22 Schematic of the simple confocal microscope that produced much of the data not otherwise referenced in this text. Specific examples of components are given for a configuration to measure diffusion spFRET or FCS (autocorrelation or cross-correlation) for the dye pair Alexa Fluor 488 and Alexa Fluor 594 (Moelcular Probes Inc., USA).These examples represent, in most cases, an arbitrary choice of supplier and are provided only to enable the user to see the specifications of the parts used. (Figure was kindly produced using Zemax (Zemax Development Corporation) by Kurt Baldwin at Avacta Ltd www.avacta.com).

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.

Deniz etal. [82 ] applied spFRET in one of the first studies that demonstrated its use as a potential structural probe revealing heterogeneity of proteins in solution. Chymotrypsin inhibitor 2 (CI2) was FRET labelled with the dye pair TMR (tetramethylrhodamine) and Cy5 (see Chapter 4). Single molecule diffusion experiments were then performed as a function of chemical denaturant. The folded and unfolded subpopulations of the protein at various denaturant concentrations were resolved and the populations of each state (estimated by the relative area of each peak) were shown to be in broad agreement with the supporting ensemble measurements. Further, changes were seen in the mean FRET efficiency of the unfolded distribution with increasing denaturant. 

---