Motor Proteins

1. Molecular Proteins at work for nanotechnology[Science 2007]

2. Actin-based metallic nanowires as bio-nanotransporters[Nature Materials 2000]

3. Powering an inorganic nanodevice with a biomolecular motor [Science 2000]

Living Cell – Miniature Factory with Protein Machine

• Create a full copy of itself in less than an hour

• Proof-read and repair errors in its own DNA

• Sense its environment and respond to it• Sense its environment and respond to it

• Change its shape and morphology

• Obtain energy from photosynthesis of metabolism just like solar cells & batteries

• Thousands of sophisticated proteins optimized by billions of years of evolution

Motor Proteins in a Cell

Molecular Motors

• Skeletal Motors– Myosin: muscle contraction

– Kinesin: transport cargo from the nucleus along microtubules

– Dynein: transport carbon towards the cell nucleus along microtubules

• Polymerization Motors– Actin

– Microtubule

• Rotary motors– ATP synthase

• Nucleic acid motors– RNA/DNA polymerase

Motor Proteins

• Motor proteinsMotor proteinsMotor proteinsMotor proteins convert the chemical energy contained in adenosine-5’-triphosphate (ATP) into the mechanical energy of movement: Myosin, Myosin, Myosin, Myosin, KinesinKinesinKinesinKinesin, , , , energy of movement: Myosin, Myosin, Myosin, Myosin, KinesinKinesinKinesinKinesin, , , , and and and and DyneinDyneinDyneinDynein

~ 7.3 kcal/mol~ 7.3 kcal/mol~ 7.3 kcal/mol~ 7.3 kcal/mol

ATP

Muscle

wiki

Sliding Filament Model of Muscle Contraction

Myosin

• MyosinMyosinMyosinMyosin is a contractile protein.

• Each myosin molecule is composed of two polypeptide chains twisted together.

• One end of each is folded into a globular head called the myosin head or myosin cross-bridge. myosin head or myosin cross-bridge.

• In the presence of calcium ions, the heads interact with specific sites on the thinner actin filaments.

• The cross-bridges contain ATPase and generate the tension developed by a muscle fibre when it contracts

Motility cycles of Muscle Myosin

• Two identical heads

– Catalytic core : blue

– Lever arms : yellow

• Two myosin heads act independently and only one attaches to actin at a time

1. Catalytic core binds weakly to actin

ADP-Pi

Pi

1

2

2. One head docks properly on the an actin binding site (green)

3. Docking causes phosphate release and lever arm swings and

move by 10 nm

4. ADP dissociates and ATP binds and reverts to its weak-

binding actin state

ADP

ATP

3

4

Kinesin and Dynein

1.1.1.1. Kinesins Kinesins Kinesins Kinesins move on the much larger microtubulepowered by the hydrolysis of ATP. They achieve this probably by sequentially swinging their head domains to the front. The active movement of kinesins pulls attached cargo through the viscous cytoplasm to deliver it to the required location. Kinesins move toward the microtubules' plus end, Kinesins move toward the microtubules' plus end, are called plus-end directed motors towards the periphery of cells (mitosis, meiosis, mRNA and protein transportation)

2.2.2.2. DyneinDyneinDyneinDynein transports various cellular cargo by "walking" along cytoskeletal microtubules towards the minus-end of the microtubule, which is usually oriented towards the cell center. Thus, they are called "minus-end directed motors"

Kinesin and Dynein

Microtubules

• One components of cytoskeleton– Diameter: ~25 nm

– Length: 200 nm ~ 25 µm

• Polymers • Polymers – α- and β-tubulin dimers protofilament

– 13 profilaments Microtubules

• Polarity– (+) end: β-subunits (elongation occurs here)

– (-) end: α-subunits

Formation of Microtubules

Disassembling Microtubule

Motility Cycle of KinesinCore (blue) is bound to microtubule. Neck linker points forward on the trailing head (orange) and rearward on the leading head (red). ATP bindingto leading head initiate neck linker docking

Neck linker docking is completed by the leading head (yellow). Throws the partner head forward

Tubulin heterodimerGreen: beta-subunitWhite: alpha-subunit

head (yellow). Throws the partner head forward by 16 nm

After random diffusional search, new leading head docks – complete 8 nm motion of the cargo – accelerate ADP release. Trailing head hydrolyzes ATP to ADP-Pi

After ADP dissociates, ATP binds to the leading head and the neck linker begins to zipper on the core. Trailing head released Pi and is in the process of being thrown forwarded

Kinesin on a Microtubule

2. Actin-based metallic nanowires as bio-nanotransporters

G-actin (globular) and F-actin (filamentous)

Actin Polymerization

Assembly of Actin-based Au Nanowires I

G-actinActivated acids: esters with good leaving group

Direct conjugation of AuNPs is not good because AuNPs may block polymerization sites

NHS

• N-hydroxysuccinimide (NHS)– Activating reagent for carboxylic acids

– React with amines to form amides

Assembly of Actin-based Au Nanowires II

AFM: Au labeled G-actin

STEM: AuNP+G-Actin

AuNW on F-actin

Length: 2~3 µmHeight: 80-100 nm

Patterned Actin-based Au NW I(actin-Au wire-actin system)

Patterned Actin-based Au NW II(Au wire-actin-Au wire system)

1-3 µm long120 nm high

0.4-1 µm120 nm high

6 nm highPolymerization of the G-actin on (+) end of the filament is faster than polymerization of (-) end

Electrical Conductivity of an Actin-based AuNW

R = 300 Ω for d ~ 100 nm L ~ 1 µm

Vs. R = 1014 Ω for unlabelled or NP-labelled filaments

ATP-fuelled Motility of the Actin-Au nanoblock-Actin Filaments on Myosin Interface

Addition of ATP the filament started to move Addition of Proteinase K the filaments stopped moving (Proteinase K: hydrolytic enzyme that degrades proteins)

ATP-fuelled Motility of the Actin-Au nanoblock-Actin Filaments on Myosin Interface

5 s 10 s 15 s0 s

Motility rate ~250 nm/s, which is 16 fold slower than that of unlabelled actin case (~4 µm/s). This is probably due to heavy Au element

Actin-Au-Actin filaments were stabilized/polymerized by phalloidin

Phalloidin binds F-actin and prevent its depolymerization

Stretching and Transporting DNA via Kinesin

Nano Lett. 2003, 3, 1251

Transporting DNA

• Microtubles: Rhodaminelabeled (Red)

• DNA (Green)

Stretching DNA• a) Between a

microtubule and surface

• b) Between Microtubules

Changing Trajectory

3. Powering an Inorganic Nanodevice with a Biomolecular Motor

FFFF1111----ATPaseATPaseATPaseATPase: : : :

8 nm in diameter, 14 nm in length Produce 80~100 pN nm of rotary torque

+ 7.3 kcal/mol

Adenosine triphosphate (ATP) rotate

Sodium azide (NaN3) stop

Three Components

• Ni posts: engineered nanofabricated substrates

• Recombinant F1-ATP: biomolecular motor • Recombinant F1-ATP: biomolecular motor – 10 histidine tag for Ni post

– Biotin/streptavidin for nanopropellers

• Nanopropellers: coated with biotin

Streptavidin-Biotin & Histidine Tag

• Streptavidin-Biotin: One of the strongest non-covalent bonds

Dissociation Constant

AxBy xA + yBAxBy xA + yBKd = [A]x[B]y/[AxBy]

SB S + BKd = [S][B]/[SB] = 10-15 mol/L

• Histidine Tag - Immobilization of proteins on a surface such as a nickel or cobalt

F1-ATPase biomolecular motor-powered nanomechanical device

Nickel Post

h = 200 nm

d = 80 nm

F1-ATPase

Nanopropeller

l = 750 or 1400 nm

d = 150 nm

Image Sequence of Nanopropellers

A: 8.3 rps, B: 7.7 rps

Calculated torque ~ 20 pN·nm

Energy used for one complete roation ~ 120 pN·nm (efficiency ~ 80%)

Time Course of F1-ATPase γSubunit Rotation

L = 750 nm, 8.0 rps

L = 1400 nm, 1.1 rps

L = 1400 nm + NaN3

(inhibitor)

Microrotary motor powered by bacteria [PNAS 2006]

• Three main components: – Si circular track

– SiO2 rotor

– Mycoplasma mobile (M. mobile)

• M. mobile– Gliding bacteria

– Cell body ~1 µm

– Moves at 2-5 µm/s

Fabrication Processes

For cell adhesion/gliding

Mercaptoundecanoicacid

sonication breaks two bridges

SEM images of Si track and SiO2 rotor

Rotation of rotors by Bacteria

Rotate at 2 rpm