Cells are alive, dynamic structures that need to transport cargoes over large distances (up to mm!) and therefore require motors to transport these cargoes.
What's more, cells contain vast amounts of DNA (each human genome is ~2m long) and these serve as tracks for enzymes that replicate, repair and read the DNA.
What are molecular motors?
Molecular motors are enzymes that convert chemical energy (ATP) into translational movement. Examples are myosin and kinesin:
Myosin (movie from the Vale Group UCSF)
This motor is involved in a variety of roles from muscle contraction to cargo transport.
This video of muscle contraction shows how myosin II's ATPase is coupled to its ability to generate motion.
For more information on myosin click here.
Kinesin (movie from the Vale Group UCSF)
This motor is mainly involved cargo transport through the cell. This video also shows how kinesin's ATPase is coupled to its motility.
For more information on kinesin click here.
Single Molecule Enzymology
What's the difference?
None really, this is just another way of saying that we study moving proteins that don't use a nucleotide energy source to bias their motion; instead they use thermal energy and therefore move by Brownian motion in a random walk.
We are studying the motion of DNA binding proteins as they diffuse one-dimensionally along their track, DNA. The video below shows a fluorescently labelled protein as it diffuses over microns of DNA!
Our techniques
The primary technique used is called Total Internal Reflectance Fluorescence Microscopy, fortunately it has a nice acronym: TIRFM
The principle behind TIRFM is based on high-school physics, when light passes from glass to water its trajectory is bent according to Snell's law as it passes into the water in a process known as refraction.
ni.sinqi=nt.sinqt --------- Snell’s Law
When light strikes the glass-water boundary at very high angles of incidence (qi) eventually the beam emerging from the glass bends so much that it runs parallel to the boundary. This is known as the critical angle. y.
At angles above this the light is totally internally reflected:
Now for the cool bit: at the point where the light is bent back into the glass a small field of light is produced that doesn't propagate into the water. This field is known as an evanescent wave, and is great for visualizing particles close to the surface:
Using this technique we can image cell surfaces and any other structures within ~200nm of the surface.
To get TIRF we use Objective-type illumination:
Photos of the TIRF in construction
Department of Biological Sciences
Contact Details:
Neil Kad
Dept. of Biological Sciences
University of Essex
Wivenhoe Park
Colchester CO4 3SQ UK
+44 (0)1206 874403