The Forisome: a smart plant protein

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The Forisome a smart plant protein

• Amy Shen
• Washington University in St. Louis, USA

Email aqshen_at_me.wustl.edu Web site
http//www.me.wustl.edu/ME/faculty/aqshen/personal
.html
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Collaborators

• Michael Knoblauch Winfried Peters, University
  of Geisson, Germany
• William Pickard, Washington University,
  Electrical Engineering
• Rahmat Shrureshi, University of Denver
• Students Steve Warmann, Rahul Blinge
• Acknowledgement

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Outline

• Motivation biomimetic materials
• Forisomes (plant protein)?
• Comparisons between forisomes and other smart
  materials
• Forisome conformational kinetics
• Biomechanics of forisomes

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Goal

• To engineer autonomous and robust biomimetic
  smart materials that outperform the current
  smart materials.

Biomimetics is the field of materials science
that is inspired by the biological systems in
nature for the design of novel materials. The
materials and structures involved in natural
systems have the capacity to sense their
environment, process this data, and respond.
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The phloem is a microfluidics system, in which
mass flow is driven by gradients of hydrostatic
pressure (up to 2 MPa)?
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FORISOMES
foris (latin) the wing of a gate or door
soma (greek) a body
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Forisomes are cellular stopcocks that reversibly
shut down individual sieve tubes
They might provide a versatile defense mechanism
against phloem-feeders
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Stopcock mechanism

• Elongate protein bodies, which we have called
  forisomes (gate-bodies), block individual sieve
  tubes in response to increased cytosolic
  Ca2-concentrations. Forisomes are thought to be
  comprised of three proteins, somewhat similar to
  a cell.

• SE Sieve Elements
• SP Sieve Plates
• PC P-protein Crystalloid
• DPC Dispersed P-Protein Crystalloid
• CC Companion Cell
• N Nucleus
• V Vacuole
• C Chloroplasts
• M Mitochondria
• ER Endoplasmic Reticulum
• PP Parietal P-proteins
• Pl Sieve Element Plastids

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Some observations of forisomes

• Forisomes are micron sized aggregations of
  proteins that respond within 50 ms to
  concentration variations of the calcium ion and
  pH.
• Forisomes perform an anisotropic change of shape
  during which their volume increases more than
  three-fold. This process is independent of ATP,
  and is driven by the binding of Ca2 (or change
  of pH) to the protein matrix. It is fully
  reversible (swell and shrink) on a similar
  time-scale by removal of Ca2, and can be induced
  electrically in vitro.

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Forisomes are contractile
.... and they exert substantial force
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Current research focus

• To acquire a basic knowledge of the detailed
  mechanisms underlying forisome dynamical
  behaviors to lay the fundamental ground for
• Synthesis of forisome based smart materials
  combined with genetic engineering.
• Forisome based valves, actuators inside small
  scale devices
• Specific tasks
• Forisome conformation and actuation kinetics
• Biomechanics of forisomes (force measurement,
  energy density, etc)?
• Biomimetic microfluidic system for valves/sensors

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Forisome conformation kinetics

• Conformation change in a forisome offers a method
  by which a plant quickly suspends mass flow of
  sap to an injured sieve element.  Using the
  forisome for similar functions in engineering
  applications demands fast response times.
• Forisomes showed average response times in the
  100 millisecond range.
• Gain insight to structural makeup (different
  response to different stimuli)?
• Understand the differences between various
  forisome species (Canavalia, soy, vicia faba)?
• Characterize the speed and geometry for
  engineering applications

Photron PCI 1280 fast cam, 10,000 fps
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Canavalia forisome with tails
Condensed state Ca 0 Dispersed
state Ca 10 mM Length of bar 25 µm
Dispersed state Ca 10 mM
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Soy forisome increase pH to 10.5
10mM EDTA, 100 mM KCl, 10 mM Tris buffer Taken
2000 fps, playing at 30 fps
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Lower pH from 10.5 to 7.5
Add sodium sulfuat, HEPES Tris, etc to adjust
calcium, pH.
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Forisome actuation dynamics
Removing Calcium concentration
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Reversibility
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Observations of forisome kinetics

• Both forisome length and diameter showed a
  biphasic pattern an initial phase of rapid
  change followed by a phase of slower change.
• The soybean forisome reduces in length by roughly
  1/3 and increases in width 2.5- to 3-fold in
  response to calcium ions. This corresponds to a
  calcium-dependend volume increase by a factor of
  5 to 6. During the reaction, the two tips of the
  forisome move with respect to each other at
  velocities of up to 40 µm per second or more,
  corresponding to 6 times its own length per
  second. The 10-90 response time in terms of
  volume (maybe the most meaningful geometric
  parameter) averaged 130-140 msec for the calcium
  response, and 105-110 msec for the chelator (pH)
  response.

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Prospective Energy Densities of forisomes

• Forisomes could become an important smart
  material if the energy density of transformation
  exceeds 0.5 MJ m3. With the zipper transition
  sequence, it is possible to achieve this if the
  modules in the crystal are roughly 10 nm on a
  side.
• No ribbon diagrams for forisome proteins means
  no robust estimates for Energy Density. However
  
• When calcium is released into cells, it can
  interact with calcium sensing proteins and
  trigger different biological effects, causing a
  muscle to contract.
• Calmodulin acts as an intermediary protein that
  senses calcium levels and relays signals to
  various calcium-sensitive enzymes, ion channels
  and other proteins. Calmodulin is a small
  dumbbell-shaped protein composed of two globular
  domains connected together by a flexible linker.

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Ribbon diagram of calmodulin with bound calcium

• The calcium ions are shown in purple. The
  calcium-binding motif is comprised of a
  characteristic loop flanked by two alpha
  helices. As shown on the right, the
  positively-charged calcium ion is surrounded in
  the loop by negatively-charged sidechains of
  three aspartates and one glutamate, as well as
  one oxygen atom from the backbone of the protein
  chain.

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what happens at a single calcium binding-site?

Acidic (negative)?
Basic (positive)?
Partially Unzipped
Zipped
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Our energy density derivation is consistent with
the Large volume change during conformation change

• Structural rearrangements at nano and meso levels
  are constrained by a loose requirement for local
  electroneutrality. Hence, between two repelling
  basic regions on adjacent fibrils, there will be
  attracted at least two anions plus water of
  hydration for the four charged moieties.
• Effective fibril radius might increase roughly
  0.65 nm due to hydration of binding sites and
  roughly 0.88 nm due to attracting hydrated
  counterions.
• If the fibrils of the crystalloid eventually turn
  out to be of small diameter (3 nm), hydration
  and counter-ion binding could double the
  effective fibril diameter and swell the forisome
  volume four-fold.

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Biomechanical testing

• Biomechanical testing is performed to show that
  the mechanical properties of the forisome are
  desirable for potential engineering applications.
  
• Tensile tests show that the mechanical
  properties are indicative of a porous structure
  with highly aligned fibers. Initial estimates
  for the Youngs modulus in the linear region of
  the sword bean forisome averages about 0.1 GPa.
  More detailed calibration is being performed for
  a more accurate estimate.

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Force measurements

• Measure forces produced by the forisome in
  swelling and deswelling and its repeatability
• To suggest maximum forces producible with
  forisomes

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Forisomes inside microdevices

• Forisome surface binding properties tethering on
  different surfaces (hydrophobic and hydrophilic).
  In order to eventually create a composite smart
  material with the forisome, it will be necessary
  to find a material which the forisome binds well
  to.
• We will utilize microfluidic device to study
  forisome behavior.

• Small reagent volumes
• Multiple forms of analysis (lab on chip)?
• Inexpensive and easily reproducible
• PDMS is gas permeable

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Forisome suspension inside a T-channel

• By adjusting the flow rates of forisome
  suspension and the calcium solution, we can
  effectively control the calcium release rate to
  contact the forisomes.

10 mM Calcium V-media (lt 0.25 mL/hr)?
10 mM EDTA V-media with forisomes (1.5--3.0
mL/hr)?
Study calcium effect on the forisome conformation
kinetics
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Forisomes binding with substrates

• Forisomes bind very well to glass and easily
  attached to the glass pipette.
• Forisomes can be easily molded without damaging.
• Inside a confined geometry, it is necessary for
  the forisomes to bind to the glass in order to
  stay in the channel, this adhesion has adverse
  effects when trying to swell and deswell the
  forisomes. When a forisome binds entirely to the
  glass, it no longer exhibits the contraction and
  expansion displayed by forisomes in other
  environment. This situation is somewhat analogous
  to fixing a piece of elastic to a plank of wood.
  Under these circumstances, the piece of elastic
  will no longer stretch and remains fixed to the
  wood.

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Observations

• The contraction of a forisome within the channels
  is observed to occur in two distinct fashions-
  one, the forisome is bound at both ends to the
  glass and appears to shimmer or wiggle or, the
  forisome is bound at one end to the glass, and
  the rest of the forisome contracts towards that
  point.
• The microchannels seem to be a superior method
  for viewing and manipulating the forisomes.
  Calcium solution can be instantly introduced to
  and removed from the forisome. This can also be
  done reproducibly and indeed over 500 repetitions
  of swelling and de-swelling were observed with a
  single forisome in less than 30 minutes.

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Increase in width of forisome vs. Flow rate of
calcium solution
Increase in Width
Flow Rate of Calcium Solution (ml/hr.)?
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Decrease in Length vs. Flow rate of calcium
solution
Decrease in Length
Flow Rate of Calcium Solution (ml/hr.)?
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Conclusions

• Forisomes are protein aggregations that respond
  within milliseconds to concentration variations
  of the calcium ion, pH.
• With calcium ion concentration as the stimulus,
  it has the all or none feature.
• Forisomes are able to swell and contract
  reversibly at high speed in two orthogonal
  directions anisotropically.
• The properties of reversibility and the speed of
  conformation action make the forisomes ideal
  candidates for development as novel biomimetic,
  synthetic machines.
• Conformational kinetics and materials
  characterizations of forisomes have been studied.

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Ongoing work

• Forisome smart fluids
• Triggering system
• Modeling

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Forisome motors
Two vicia forisomes held by microneedles,
attached to a micron size glass bead
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Self-powered sensory nerve system

• To design a self-powered structural monitoring
  and diagnostic system that mimics the sensory
  nerve system of a human body, by utilizing a
  novel, non-living plant protein (forisome) for
  sensing and information transfer. (With Rahmat
  Shoureshi)?