Chemistry Reference
In-Depth Information
diffusion. The motion of single molecules on the cell surface and inside the cell has
also been measured using
fluorescence correlation spectroscopy (FCS) [19, 20].
The interaction between ligands and receptors has been studied using single
molecule imaging and mechanical measurements (see Chapter 7). The binding of
ligands to receptors triggers signal transduction processes leading to the activation of
whole cells (see Chapter 5). The activation of a cell can be directly related to the binding
events of the ligands inside the cell. Such an experiment was
first carried out by
increasing the cytoplasmic Ca
2 รพ
concentration to determine the number of peptide-
MHC ligands bound to a T-cell [21]. In general, only a small number of binding ligands
have been reported in various systems. In fact, results have emphasized that small
input signals within large noisy signals can generate consistent output. For example,
indictyosteriumcells, inwhichmotion is biased in one direction in response to a small
cAMP concentration gradient, the kinetics of cAMP binding was observed to be
dependent on location, leading to the polarity of the moving cell [22].
The expression of RNA and protein has also been detected in live cells at the single
molecule level. RNA plays a pivotal role in gene expression in which RNA molecules
work dynamically in the nucleus and cytoplasm (see Chapter 8). The traf
cking of
messenger RNAwas monitored in real time after being expressed inside the nucleus
of live cells [23]. It was demonstrated that diffusion is the basis for mRNA displace-
ment. Regarding proteins, individual proteins expressed in single cells have been
counted in real time. YFP
fluorescence showed [24] that protein expression occurs in
bursts. The stochastic nature of the gene expression has also been studied.
1.4
Fluorescence Spectroscopy and Biomolecular Dynamics
Combined with single molecule imaging,
uorescence spectroscopy can be used
to measure structural dynamics and biomolecule interactions in real time (see
Chapter 9). In particular,
fluorescence resonance energy transfer (FRET) between
single
fluorophores has become a popular tool of choice following its application in
dried DNA using scanning near-
eld microscopy [25]. Conformational dynamics
were observed using chymotrypsin inhibitor 2 [26] and Tetrahymena thermophila
ribozyme [27]. These conformational studies have been followed by a large number of
studies on multiple conformations and rugged energy landscapes. Single molecule
FRET has also been used for protein folding studies. Subpopulations of folded and
denatured conformations of proteins freely diffusing in solution were directly
determined by confocal microscopy [28, 29]. These measurements have provided
useful tests for a new view on dynamic structures of biomolecules in which they
behave according to the energy landscape. In addition to FRET, spectral shift and
fluorescence lifetime polarizations have been used to study protein dynamics. Single
molecule
first carried out to monitor the axial
rotation of actin
filaments sliding over myosin immobilized on a glass surface [30].
It was recently demonstrated using polarization imaging from
uorescence dyes
attached bifunctionally to the neck domain of myosin V that the orientation of the
fluorescence polarization imaging was
