In addition to its impact on small molecule motion, confinement can also impact motion of macromolecules. Thus, we will investigate both overall macromolecular motion and local motion of parts of the macromolecules. We propose to investigate these effects in the timescale range of sub-ms to seconds, the critical timescale for key events in cell membranes and in the cytoplasm. In this manner the results in this aim will provide fundamental information which when the location of these probes enhance interpretation for experiments described in Project 4. Three general classes of intramolecular processes will be studied: photoisomerization and back-isomerization of a small organic dye molecule, the rotational motion of fluorescent probe molecules confined in phospholipid bilayers, and folding and unfolding of structured DNA hairpins. The photo-isomerization and DNA folding reactions will be studied in bulk solvent and reverse micelles using fluorescence fluctuation spectroscopy. Fluorescence and phosphorescence lifetime measurements will measure intramolecular movement allowing comparison to corresponding chromophores free in solution. Together, these experiments probe the effects of restricted environments on intramolecular motion in a simple and well-defined molecules, in a molecules with greater flexibility and finally in a system of biological significance.
Figure:
Fluorescence correlation spectroscopy (FCS) is an extremely
useful technique in that it allows one to study
the kinetics of a chemical or biological process without disrupting the
system’s thermal equilibrium. This is accomplished by recording and
analyzing fluctuations in the fluorescence signal due to the process
under study. In autocorrelation methods, the recorded fluorescence
signal is continuously and compared with itself at
some time later (lag time). From these data, an autocorrelation
function is calculated containing kinetic as well as diffusional
information about the process/molecule under study may be calculated.
One goal of our research is to study the behavior of biomolecules when they are confined in reverse micelles. As a first step, we have placed the dye molecule we intend to use (Rhodamine 6G or R6G) inside reverse micelles in order to determine if FCS is feasible. This figure shows autocorrelation functions taken from different sized reverse micelles that contain R6G. These data indicate that FCS is indeed capable of monitoring molecules inside reverse micelles.