Case Studies
iXon EMCCD Camera Helps in Discovering Secrets Behind Protein Unfolding
Dr. Atom Sarkar, is a neurosurgery resident at the Mayo Clinic (Rochester, MN) but
is carrying out his research goals in Professor Julio Fernandez’s Single Molecule
Force Spectroscopy lab at Columbia University (New York, NY). The role of nanotechnology
in neurosurgery is central to his research.
Currently he is delving into the world of protein mechanics. Why? Because a knowledge
of a protein’s structural dynamics is central to the understanding of many neurological
diseases ranging from brain tumors to neurodegenerative processes such as Alzheimer’s
Disease. Protein folding and unfolding happen on the nanometer scale. Therefore
Atom needs a technique of nanometer or sub-nanometer resolution. Confocal won’t
cut it . He has been using a customized total internal reflection (TIRF)/atomic
force microscope (AFM)/electron multiplying charge coupled device (EMCCD) to study
protein unfolding, with the aim of developing a TIRF-only method to follow protein
dynamics with sub-nanometer resolution. This will open protein unfolding experiments
to many more scientists.
The key principle behind the methodology is the use a nanometer-scale calibrated
evanescent wave to measure the position of a fluorophore moving along the optical
axis. By using a TIRF generated evanescent wave that has an intensity which decays
exponentially as a function of vertical distance, Atom can correlate changes from
fluorescence intensity into displacements in the vertical axis.
In order to calibrate the evanescent wave, an AFM/TIRF/EMCCD device was constructed
consisting of an AFM head mounted on top of a TIRF microscope equipped with an electron
multiplying charge coupled device (EMCCD) (see figure above). The system exploits
the distance dependent evanescent wave as a ruler
to deconvolve fluorescent
intensity into length (see figure below).
The six curves depicted in the graph above nicely illustrate the measured evanescent
field decay and how by changing the TIRF conditions, the evanescent wave penetration
depth, dp, can be adjusted to fit experimental needs. The ability to measure distance
with the evanescent field has been dubbed Evanescent Nanometry.
Evanescent Nanometry has already been used to follow the forced mediated unfolding
of the protein ubiquitin, and these results rival the precision and accuracy obtained
by standard AFM only studies.
Current experiments involve replacing the AFM entirely and attaching magnetic fluorescent
beads to the proteins. The magnetic beads allow the tethered proteins to be manipulated
by an electromagnetic tweezer set-up, while the fluorescence of the bead enable
nanometer precision monitoring of the proteins unfolding and folding dynamics, which
are captured by the EMCCD.