Biomedical Engineering Reference
In-Depth Information
time frame associated with excited-state processes. In 1994, Trautman et al. (79) reported
that the emission spectra of single molecules dispersed on a film of PMMA changed with
time. In the same year, Ambrose et al. and Xie et al. (80,81) each reported the observation
that there were sudden jumps in the emission intensity from single rhodamine molecules
dispersed on a glass surface. Furthermore, Xie et al. modulated the polarization of excita-
tion light to examine whether the single-molecule spectral jumps were due to molecular
reorientation on the surface. This represented the beginning of direct monitoring of
dynamics of single molecules (15). Many other experiments have subsequently been
reported and cover topics such as the Brownian motion of single chromophores in a lipid
membrane (3) and spectral fluctuations of single sulforhodamine-101 molecules (82,83).
Radiative and nonradiative energy transfer, charge transfer, electron transfer, and other
processes have been studied by single molecule detection (15). Lu and Xie (84) reported
the single-molecule chemical kinetics for interfacial electron transfer from cresyl violet
molecule to indium tinoxide nanoparticles. Importantly, it was demonstrated that electron
transfer data obtained from a single molecule detection system showed single exponential
kinetics, compared with the observation of multiexponential kinetics from an ensemble
average of experiments using the same chemical system. The reason for this contradiction
lies in site heterogeneity of single dye molecules with their environments.
2.4.5
Single Molecule Detection in Biomolecular Dynamics
Conformational states and changes between states are fundamental to most biochemical
processes that play a role in biological function. Often there are many states and many con-
formations that are weakly separated in energy. Such states cannot be easily distinguished
by ensemble-average experiments. Single molecule detection techniques have been used in
a broad range of applications in life sciences and biotechnology, including molecular
motors, DNA transcription, enzyme reactions, protein dynamics, and cell signaling.
Individual enzyme reaction activities based on different conformational states is an area
of substantial interest. A review by Greulic (85) reports techniques for determination of the
activity of a single enzyme molecule. Xue and Yeung (86) have confined single lactate
dehydrogenase molecules along a capillary to detect individual molecular activities, and
the catalytic activities among different enzymes have been found to vary by up to a factor
of four. This result suggests the presence of different enzyme conformational states.
Fluorescence resonant energy transfer has been used to observe ribozyme folding inter-
mediates (71). Kinosita and coworkers (87) have observed the discrete and stochastic 120º
rotation steps of the g subunit in F1-ATPase. The 120º rotation step is composed of a 90º
rotation step corresponding to the binding of ATP and a 30º rotation step corresponding
to the hydrolysis of ATP.
Using single molecule detection technique, it is possible to probe the heterogeneity in
reaction rates among nearly identical molecules, and also to study the extent to which
activity is related to the previous reaction history. Such a “molecular memory effect” has
been observed for flavoenzyme when it catalyzes the oxidation of cholesterol by oxygen
(88,89). This experiment was done using a confocal microscope with enzymes immobi-
lized in an agarose gel. This experiment provided evidence that the memory effect was
due to slow conformational fluctuations of the enzyme molecules.
Besides enzyme reactivity studies, single molecule detection method has also been
widely used in the area of protein dynamics and protein folding. The fluorescence of GFP
from the jellyfish
Aequorea victoria
shows repeated on/off cycles on a time scale of seconds
(90). Wazawa et al. (91) also observe similar spectral fluctuation of a single fluorophore,
tetramethylrhodamine conjugated to the myosin S1, on the same time scale. The
