The kinetics of proteins on lipid bilayers
Date
2017
Authors
Nepal, Kanti, author
Krapf, Diego, advisor
Peersen, Olve, committee member
Levinger, Nancy, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Signaling molecules trigger downstream signaling pathways when they arrive at the plasma membrane. They have to be recruited to the plasma membrane by membrane targeting domains. Our experiments throughout focus on understanding kinetics of C2 domain's diffusion on the membrane. In contrast to trans-membrane proteins, interactions between these domains and the plasma membrane is found to be peripheral and transient. These proteins perform two dimensional diffusion on membrane surfaces and faster three dimensional diffusion in the bulk. We label proteins at the single molecule level and do single particle tracking. In addition to two dimensional surface diffusion, it is sometimes observed that they dissociate from the membrane and rebind at a another location of the membrane after a short journey in the bulk solution. The time averaged mean square displacement (MSD) analysis of individual trajectories is linear whereas ensemble average MSD is superdiffusive. The distribution of displacements fit to a Gaussian distribution followed by a long tail which is Cauchy's distribution. This long tail in cauchy's distribution is from the larger displacements caused by jumps of molecule to explore greater area for efficient target search. The second section of this thesis explored the effect of crowding agents on these proteins. Polyethylene glycol (PEG) is used here to simulate crowded cellular environment aiming to understand its effect on membrane targeting C2 domains as well as on the lipid bilayer. In this chapter, we recognized that a crowding agent like PEG plays a significant role in changing the trend on diffusion behavior of C2 domains. When the PEG concentration is increased, there is a decrease in the transition of molecule between the surface and the bulk phase. With the same series of PEG concentration, there is increase in population of immobile C2 domains and desorption time. But no such increasing or decreasing trend is seen on the lipid bilayer alone. Experiments were reproduced and imaged a number of times using total internal reflection (TIRF) and fluorescence recovery after photobleaching (FRAP) techniques. Lastly, a small part of my thesis also dealt with set of experiments done to monitor tethered particle motion of DNA as well as flow extension experiments on DNA and RNA using bright field microscopy. DNA/RNA had beads tethered to one end of the strand and other end to the cover glass. Primary results are presented.