Immobilized single molecules measurements

Abstract. Various ways of extracting information on the conformational structure, dynamics and correlations between them from single-molecule measurements of florescence resonance energy transfer are surveyed. The information obtained via those various ways is then analyzed in detail in the case of an off-lattice model of a two-stranded coiled-coil polypeptide that follows Langevin dynamics. The analysis includes a consideration of the cases of a freely diffusing and surface-immobilized polypeptide as well as the effect of different types of surface and denaturation conditions. 

In this section we outline the two main types of single molecule measurement that we have chosen to discuss in detail in this text measurements on diffusing fluorescent single molecules and measurements on immobilized single fluorescent molecules. We introduce the basic concepts of these experiments, which we then expand upon in both a phenomenological and rigorous mathematical way in subsequent chapters. 

Figure 1.2 Illustration of the concept of measuring the fluorescence from an immobilized single molecule, (a) A molecule, which can undergo a reversible transition between folded and unfolded conformations, is labelled with a dye at one terminus and a quencher at the other. In the folded (native) conformation the fluorescence from the dye is quenched. In the unfolded (denatured) conformation the fluorescence is enhanced. (b)The molecule is immobilized (tethered) onto a solid substrate and the fluorescence signal from a small volume near the surface monitored as a function of time. (c)The same molecule can be monitored for a considerable length of time and the stochastic transitions (the number of which depend on the height of the energy barrier for the transition) can be obsen/ed. Eventually (at around 9.5 s in this simulated example), photobleaching of the dye occurs to a non-fluorescent state, at which point no more information can be extracted from this molecule.

In AFM single molecule dynamic force spectroscopy (AFM-FS), binding forces between two molecules of interest are measured by functionally immobilizing them on two different surfaces (i.e. the tip of an AFM and an appropriate counter surface) that are brought in mechanical contact under physiological conditions (see Figure 2). 

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