nanobiology enzymes as nanomachines molecular motors fluctuations in gene expression fluctuations in...

NanobiologyNanobiology

• Enzymes as nanomachines

• Molecular motors

• Fluctuations in gene expression

• Fluctuations in gene splicing

Copyright Stuart Lindsay 2009

• The genetic material (DNA) is contained inside the nucleus.

• Genetic information is transported to the cytoplasm as an RNA copy.

• ribosomes translate the RNA code to proteins

• the entire cell is packaged in a lipid bilayer membrane.


The Cell MachineThe Cell Machine

Proteins: the nanomachinesProteins: the nanomachines

(A) AP1: Transcription factor binding DNA to regulate the expression of genes (DNA double helix in green)

(B)Protein kinase: a dimer that phosphorylates a target

(C) Actin, a filamentous protein acting as a structural scaffold.

Copyright Stuart Lindsay 2009The 20 amino acid building-blocksThe 20 amino acid building-blocks

Genes code for amino acidsGenes code for amino acids

The genetic code: Note the stop codons and degeneracy.

Note that 42=16 (<20), 43=64 (>20), so a three bases code is necessary to specify the 20 different amino acids.

The sequence of residues is specified by the sequence of basis in RNA.

Each residue is codified by a sequence of 3 bases (codon).


The cell machineryThe cell machinery

Into the nucleusInto the nucleus

out to the cytoplasmout to the cytoplasm

trRNA

Mechanical properties of proteinsMechanical properties of proteins

StiffnessStiffness

StressStress =Force per unit area

EA

F StrainStrain =

Fractional change in dimension

Young’s modulusYoung’s modulus

Young’s modulusYoung’s modulus: ratio of stress to strain for a given material.

In terms of a Hookean spring (A=l2): ElF Spring constant: k = El

Ex. Tubulin (transport in the cell) MW=50KDa, E=2GPa, r=2.4nm, k = 10 N·m-1

Ex. Elastin (muscle and ligaments) MW=75KDa, E=0.002GPa, r=2.75nm, k = 0.01 N·m-1

DensityDensity

Typical density of a well-packed protein is ρ ≈1.4·103 kg·m-3.

nmMW.MW

r 33 0708224

3

A (typical) 100kDa protein has a radius of ~3nm.

ViscosityViscosity

Typical viscosity for a globular protein (MW=100kD; r=3nm) isη ≈1·10-3 Pa·s.

DiffusionDiffusion

aTk

D B

6

Typical diffusion coefficient is D ≈7.3·10-11 m2·s-1.

1121056 6 NsmrDrag coefficient in water:

Protein motionsProtein motions

a6

Dt2

Damped elastic fluctuations: from ps (tubulin) to ns (elastin)

Diffusive fluctuations: tens of ns

Actual catalyzed ET rate is kHz, so only ca. 1 in 106 long-range fluctuations drive the system to the transition state.

Long range, collective fluctuations:


Voltage gated channelVoltage gated channelLiving cells exchange materials by means of channels proteins,

chemically selective and under the control of signaling mechanisms.

AFM images of a monolayer of voltage-gated Porin OmpF on a graphite surface in an electrolyte solution.

The switch from open to closed is driven by the potential gradient across the cell membrane.

Energy for Molecular Motors and Energy for Molecular Motors and ATP-dependent enzymesATP-dependent enzymes

ATP HydrolysisATP Hydrolysis

5109.4][

]][[x

ATP

PADPK ieq


Thermal ratchet driven by ATP hydrolysisThermal ratchet driven by ATP hydrolysis


Reactants +ATP

Products + ADP

kT

Eexp

ak a

6

Reactants = protein motor + H2O

Products = protein motor one step forward

Molecular motors in muscle cellsMolecular motors in muscle cells

Motor function in muscle cells is carried out by an actin-myosin complex.

• Sarcomeres Sarcomeres appear as ca. 100,000 bands in cardiac muscle (TEM image)

The active components of muscle tissues are the sarcomerssarcomers, thick filaments to which are attached many myosin molecules.

Sarcomers, thick filaments, are interdigitated with thin filaments, composed of bundles of the actin protein.

Crossbridge modelCrossbridge model

Myosin molecules consist of a long stalk that is permanently attached to the tick filament and a pair of head units that transiently contact the actin filaments.

The myosin moves along the interleaved actin filaments to draw the crossbridge together, resulting in muscle contraction.

Muscle tissues can contract by more than 20% in length on a period of tens of milliseconds.

Myosin-actin motor motionMyosin-actin motor motion

Actin FilamentActin Filament

“Walking” action: step is 5 nm (amplified by lever arm to 36 nm). Force is 1.5 pN.

Arrows point to heads

Crystal structure of head unit. Note two “feet”.

ATP binding, hydrolysis and head motion

Myosin motorMyosin motor


(AAAS)

Myosin molecule

A Rotary Motor – ATP SynthaseA Rotary Motor – ATP Synthase

• Proton gradient drives F1 rotation accompanied by ATP synthesis from ADP.

• High ATP concentration drives rotation in opposite direction with ATP hydrolysis which pumps protons.


A reversible motor:A reversible motor:


clockwise rotationclockwise rotation counter-clockwise rotationcounter-clockwise rotation

Watching ATP synthase at workWatching ATP synthase at work

http://www.k2.phys.waseda.ac.jp/Researc.html

F1 unit tethered with dye-loaded actin attached to F0

(F1Prop4C.gif)


(Courtesy of Professor Kazuhiko Kinosita, Waseda University.)

Movement of the gold bead was detected by laser optical imaging.

The motor takes three 120° steps to complete one rotation, hydrolizing one ATP molecule per step.


Helix repeat: 3.4 nmHelix repeat: 3.4 nm

DNA NanotechnologyDNA Nanotechnology


About 8 bases must be paired for a double helix to be stable at room Temperature.


A DNA-based four-way crossover structures producing a rigid planar tile. The distance between adjacent tile is 20nm.The structure, imaged by AFM, is produced by spontaneous self-assembly of the individual crosses.

DNA OrigamiDNA Origami

A long template strand is annealed with a numebr of short strands that either form cross-links at fixed points (loops) or fill regions to form double helices.

Biomimetic nanostructuresBiomimetic nanostructures

MineralizationMineralization

These structures consist of

mineral layers held

together with proteins that

acts as surface-specific

‘glues’ (SEM images).

Abalone shell Diatom


Peptide glues for specific surfacesPeptide glues for specific surfaces

• Make a random peptide library on the surface of a phage by inserting random DNA into phage genome (Phage display)

• Select those phage that stick and grow them up.

• Repeat cycle for highly specific interaction

• Sequence “successful” phage genome to decode peptide sequence

Filamentous bacteriophage sticking to an InP (100) surface. They express a surface protein that sticks to just this particular surface.

Whaley, S.R. et al. Nature,2000, 405: 665-668.

http://www.nature.com/

Mimicking Bio-nano-opticsMimicking Bio-nano-optics

Brittlestar nanolenses

Lenses modeled

mimicking brittlestar

but flexible so the

refractive index can

be adjusted by pumping

different fluid.

Lee and Szema, Science 2005

nanobiology enzymes as nanomachines molecular motors fluctuations in gene expression fluctuations in...

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