Molecular Conformational Dynamics
Many of the intracellular biological processes are barely known to exist, let alone fully understood. Therefore, the primary goals of molecular and cellular biology research are not only the identification of the processes and pathways but also the understanding of the biophysical properties of the fundamental elements, e.g. oligonucleotides and proteins, in a biological system and their dynamic interactions with their surrounding environment. In order to achieve this goal, it requires techniques that are able to monitor and record these elementary interactions with both extremely high resolution and sensitivity at the level of single molecule. Only by sampling the fluctuations of a system molecule-by-molecule, it no longer measures just the average behavior but can study the full distribution of molecular function. When examining a biological system at the level of its individual components (in this case, molecules), it also leads to a deeper insight into many of the problems tackled by science today and provides an unambiguous way of distinguishing between heterogeneous and homogeneous distributions of molecular properties.
Fluorescence Correlation Spectroscopy
Fluorescence correlation spectroscopy is one of the analysis methods for studying extremely low concentrated biomolecules with high spatial and temporal resolution. In contrast to other fluorescence techniques, the primary interest for FCS is not the measurement of fluorescence intensity itself, but rather the spontaneous intensity fluctuations caused by minute deviations from thermal equilibrium of the small elements in a system. In general, all physical parameters that are able to produce fluorescence fluctuations are accessible by FCS. For example, as the fluctuations provides information on the rate of diffusion and a particle that, in turn, is directly dependent on the particle?s size. As a consequence, any increase in the size of a biomolecule, e.g. as a result of an interaction with a second molecule, is readily measured as an increase in the particle?s diffusion time. FCS, therefore, is an ideal approach to determine the thermodynamic and kinetic features such as the local concentrations, mobility, and characteristic time constants of inter- or intracellular reactions of fluorescently labeled biomolecules in a femtoliter volume.
Study of Conformational Dynamics of Biomolecules
FRET is a non-radiative transfer of energy from a fluorophore (the donor) a neighboring molecule (the acceptor), returning the donor molecule to its ground state without fluorescence emission. An excited donor molecule has several ways available to release the captured energy and return itself to the ground state, for examples, dissipating as heat to the environment or transferring to a second acceptor molecule. The efficiency of this transfer is given by
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where r is the distance between the donor molecule and the acceptor molecule , R0 is Forster distance which describes the spectral overlap and typically is on the scale of a few nanometers.
Basing on FRET, the conformational change of fluorophore-labeled molecules can be measured on the resolution of 1- 10 nm scale, which is far beyond the limit of spatial resolution of conventional optical microscopy and is ideal for studying single molecule dynamics. For example, distance changes between two sites on a biomolecule (or between two different molecules) can be real-time measured via fluorescence resonance single-pair energy transfer (spFRET) by monitoring spectral changes in the emission of a single donor-acceptor pair. Change of orientation can be also be determined via single-molecule fluorescence polarization anisotropy (smFPA) by following changes in the dipole orientation of a rigidly-attached probe. The FRET based fluorescence can be applied to explore the kinetics of biomolecules and rate constants of molecules for example DNA hybridization, gene expression pathways, protein folding, or ion channel dynamics.
