Transcript Protein-Protein Interactions in Single Bacteria Flagella

Protein-Protein Interactions
in Single Bacteria
Flagella
Aryeh Warmflash
Advisor Prof. Norbert Scherer, Department of Chemistry
Purpose
• To gain a better understanding of protein-protein
interactions in general by exploring the
mechanics of flagella
• Protein-protein interactions are crucial to
innumerable processes in biology
• There is abundant biochemical and structural
data on flagella making them ideal for study.
However, how the structure facilitates the
function of flagella is unclear.
• Studying the mechanics of flagella will help
answer this question.
Background Information
• Bacteria are propelled by tail-like appendages called
flagella
• Flagella are composed of protein subunits called flagellin
and have a helical structure which changes handedness
when the direction of rotation is reversed.
• Normal flagella are left-handed and can transition to
right-handedness. Right handed (unable to transition)
and straight mutants are also studied.
• The flagella are modeled as having two separate
interactions- soft, axial and rigid, lateral interactions
• The interplay between these is used to explain the ability
of the flagella to change states while maintaining its
overall structure.
The Model
• In the stretched state, the behavior
is elastic. We model force v.
extension as: F~k/(1-l /L) where L is
fixed contour length of the helix and
k is the intrinsic rigidity
• In the over-stretched state (when
helix is >80% extended) a rigid
linear term is added:F~k/(1-l /L) +
K(l /L-1)
• Boltzmann statistics with an
external force f then gives the
extension, l .
Method
•Flagella are covalently bonded to an Atomic Force
Microscope (AFM) Cantilever which is used to apply
a force
• Cantilever pulls with constant velocity. Force and
extension are measured
•Range is approximately 10 pN to 1 nN
Project Part I --Hysteresis
•Hysteresis has been observed in the
pulling and retracting of flagella
•Hysteresis is related to the energy
barrier between the packed and
unpacked states and the repacking rate
•In the first part of my project, I will
quantify the dependence of the
hysteresis on the rate of pulling
Project Part II – Constant Force
Measurement
• All past measurements have been done at constant
velocity and have measured force and extension.
• Part II of the project will be to design hardware which will
make the AFM cantilever pull with constant force.
• With the AFM in constant force mode, we can measure
the time intervals between breakage events as a function
of the applied force.
• This data can be used to compute a free energy curve
• This will also connect to the research on hysteresis. The
hysteresis can be used to determine the irreversible
work. Using Jarzynski’s equality, this can also be used to
compute the free energy.