The Forisome: a smart plant protein

Amy Shen

Washington University in St. Louis, USA

Email: aqshen@me.wustl.eduWeb site: http://www.me.wustl.edu/ME/faculty/aqshen/personal.html

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

Goal• To engineer autonomous and robust biomimetic smart

materials that outperform the current smart materials.

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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.

The phloem is a microfluidics system, in which mass flow is driven by gradients of hydrostatic pressure (up to 2 MPa)

FORISOMES

foris (latin):the wing of agate or door

soma (greek): a body

Forisomes are cellular stopcocks that reversibly shut down individual sieve tubes

They might provide a versatile defense mechanism against phloem-feeders

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

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.

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

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

Canavalia forisome with tails

Condensed state [Ca++] = 0 Dispersed state [Ca++] = 10 mM Length of bar: 25 µm

Dispersed state [Ca++] = 10 mM

Soy forisome: increase pH to 10.5

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10mM EDTA, 100 mM KCl, 10 mM Tris buffer10mM EDTA, 100 mM KCl, 10 mM Tris bufferTaken 2000 fps, playing at 30 fps

Lower pH from 10.5 to 7.5

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Add sodium sulfuat, HEPES Tris, etc to adjust calcium, pH.

Forisome actuation dynamics

40

30

20

10

Length (um)

0.40.30.20.1

Reaction time (s)

3500

3000

2500

2000

1500

1000

500

Volume (um

3)

Major axis minor axis Volume

Removing Calcium concentration

Reversibility

5

4

3

2

1

Length (um)

0.200.150.100.05

Reaction time (s)

Swelling when increasing pH De-swelling when lowering pH

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.

Actuation properties compared to other smart materials

lowhighlowlowlowlowEnergy dissipation

20-100 <5100K100K100>2Band width (Hz)

0.04--10030000nonlinear0.137300Young’s modulus (Mpa)

0.61000.10.020.750.3Energy density (J/cm^3)

30-20080.229607Max. strain (%)

ForisomeShape memory alloys

Piezoceramic PZT

Polyacrylate Elastomer

Electroactive Polymer

Electrostrictor P(VDF-TrFE)

Performance parameters

Prospective Energy Densities of forisomesForisomes 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 …

1. When calcium is released into cells, it can interact with calcium sensing proteins and trigger different biological effects, causing a muscle to contract.

2. 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.

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 UnzippedZipped

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.

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 Young’s 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.

Force measurements

Measure forces produced by the forisome in swelling and deswelling and its repeatability

To suggest maximum forces producible with forisomes

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

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 EDTAV-media with forisomes

(1.5--3.0 mL/hr)

10 mM Calcium V-media (< 0.25 mL/hr)

Study calcium effect on the forisome conformation kinetics

E thy le ned iami ne- T etraace tic A cid

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.

Forisome deposition dependence on flow rate

Number of Deposited Forisomes vs. Flow Rate of V-Media Containing Forisomes

0

1020

30

40

50

6070

80

0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4

Flow Rate of V-Media (ml/hr.)

Number of Deposited

Forisomes

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.

% Increase in width of forisome vs. Flow rate of calcium solution

Flow Rate of Calcium Solution (ml/hr.)

% Increase in Width

% Decrease in Length vs. Flow rate of calcium solution

Flow Rate of Calcium Solution (ml/hr.)

% Decrease in Length

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.

Ongoing work

• Forisome smart fluids• Triggering system• Modeling

Forisome motors

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Two vicia forisomes held by microneedles, attached to a micron size glass bead

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)