Bionanotechnology

Dr Cait MacPhee (cem48@cam.ac.uk)Dr Paul Barker (pdb30@cam.ac.uk)Mondays 12 pm, Tuesdays 11 am

SyllabusThe molecules of lifeProteins (6 lectures)

backgroundas components in nanodevices biomolecular electronic devices electron transport and photosynthesis as fibrous materials in motion – molecular motors

DNA (3 lectures)background as components in nanodevices: part Ias components in nanodevices: part II

Lipids (1 lecture)background; as components in nanostructures:

artificial cells (liposomes and membrane nanotubes)

Bio-inorganic composites (1 lecture)composites – including butterfly wings, diatoms,

mineralisation

The whole cell Cell mechanotransduction (1 lecture)

bringing together physical, life, and applied sciences; bone cell mechanobiology

 Cell motility (1 lecture)

how cells travel and navigate through 2- and 3 dimensional environments

 Biomaterials (1 lecture)

surface science/ surface chemistry; tissue engineering

 Nanomedicine (1 lecture)

Nanotherapeutics, real and imagined·        Qdots and developmental biology

 Ethical considerations (1 lecture)

risk/benefit analysis focusing on bio-nanotechnology

So why are we interested?

• Biological systems inherently good at self-assembly

• Multitude of functions evolved over millenia

• Bacterial expression systems allow us to artificially evolve “unnatural” functions

Protein Function

1. Structural collagen, elastin… Force transmission

2. Mobility actin/myosin, flagella… Linear/rotary motors

3. Receptors insulin receptor…

4. Ligands insulin…

5. Immune system antibodies...

6. ‘housekeeping’ chaperones... Quality control

7. signalling kinases... Amplification

8. Enzymes proteases… Chemical/ photochemical work

9. Storage and Transport haemoglobin…

Lock and key recognition

How do you produce a bucket of protein?

• Expression systems are based on the insertion of a gene into a host cell for its translation and expression into protein

• Insert the DNA coding the protein of interest into a plasmid – a small, circular piece of DNA that is found in E. coli and many other bacteria • generally remain separate from the bacterial chromosome • carry genes that can be expressed in the bacterium • generally replicate and are passed on to daughter cells along with

the chromosome

DNA RNA Protein

Protein Expression

• Results in a population of bacteria replicating many copies of a segment of foreign DNA

• Many recombinant proteins can be expressed (produced) to high levels in E. coli systems

Plus:• Unnatural amino acids

• Artificial (accelerated) evolutionO

N+

N+

O

+N

O

NH

F

F

F

NH

F

F

F

F

…..but!

• Protein sequence space is impossibly large• 2060 ~ 1078 > number of particles in the universe• More variants can be made than can be

reasonably tested– ~1015 can be made in a test tube

• can only be assayed by enrichment strategies

– ~107 can be tested with a genetic selection– ~106 -107 can be tested with an ultra high-throughput

screen• single cell assays

– 104-105 in microtiter plates– Really need to be able to screen at least 1000

samples per week for in vitro evolution

(Nanoscale) problems?

• Inertia/ viscosity; Thermal noise; Gravity; predicting sequence-structure-function relationship

• Water– The hydrophobic effect

R

–Depletion forces•Entropic effect•Large objects (i.e. proteins) are surrounded by a depletion zone of thickness equal to the radius of small particles (i.e. solutes)•Elimination of the depletion zone increases the entropy, thereby decreasing the free energy of the solute•Short range (2R) interaction

Some examples

• Viral toolkit• S-layers• HSP60• Ferritin• Bacteriorhodopsin

Viruses

• A bacteriophage is a virus capable of infecting and reproducing in bacteria

• Filamentous phage are comprised of a single-stranded DNA molecule encased in a protein cylinder

• Protein cylinder is assembled from five coat proteins

• Foreign DNA can be incorporated into the genome and “displayed” as a fusion to a coat protein

M13 phage

• Dimensions:– 6.5nm in diameter– Length dependent on genome but wild

type approximately 930nm– Mass 16.3MD of which 87% is protein

• Sophisticated life cycle: – Infection transfers genetic material into

host with little disruption.– Late infection stages perturb host

membrane for release of virions, but M13 is non-lytic. Does not kill its host – turns it into a virus factory.

DNA

g3

g8

g6

g9 g7

2,700-3,000 copies

Phage Peptide & Protein Libraries

• Libraries of peptide or protein sequences are constructed and cloned into the phage genome

•By cloning large numbers of DNA sequences into the phage, display libraries are produced with a repertoire of many billions of unique displayed proteins (i.e. a peptide of 6 amino acids = 64 million variants).

• One foreign insert per phage = a single polypeptide sequence displayed per particle

• All copies of g3 have the same polypeptide displayed (multivalency)

•Select for desired activity/ function

Selecting for non-biological functions

• Phage selected for nucleation of semiconducting CdS and ZnS particles

Single crystal nanowires

• Mineralisation: nucleation from metal salt solution at low temperatures to yield crystallographically uniform orientation of nanocrystals

• Crystals cannot fuse due to presence of nucleating peptide

• Anneal at 350°C• Successful for ZnS and CdS – single

crystal nanowires• Ferromagnetic CoPt and FePt systems

selected for development of low-dimensional magnetic materials

Mao CB, Solis DJ, Reiss BD, et al. Science 303 : 213-217 2004

S-layers

• Cover the cell surfaces of a wide range of bacteria

• Hypothesized to be involved in protection, molecular sieving, ion trapping, cell adhesion, surface recognition, and morphogenesis.

• Self-assemble into 2-D crystalline lattices in vitro, on surfaces, polymers, and liquid-air interface

• Lattices 5-10 nm thick and containing pores of 2-8 nm (depending on the species).

TEM image of a bacterial cell with an S-layer with square lattice symmetry. Bar=100nm

S-layer protein self-assembled on silicon wafer

• S-layers on solid supports nucleation and growth of CdS and Au nanoparticles in pores.

• Defined size (5 nm), defined superlattice symmetry (~13 nm spacing)

• Preformed citrate-modified gold nanoparticles self-assemble in a hexagonal array (~7000 particles/ m2, separation 10.4 nm)

• Hard disk drives currently store information at ~70 Gbit/in2; density restricted by coarse patterning/ coarse grain size and superparamagnetic effect

• S-layers potentially 4 Tbit/ in2

Bergkvist, M., Mark, SS., Yang X. et al., J Phys Chem B 108, 8241-8248 2004

S-layers

S-layers

• S-layer acts as mask to generate surface pattern in Si via plasma etching.

• 10 nm diameter magnetic dots, separation 22 nm

• No superparamagnetic effect• System is ferromagnetic at room

temperature, with magnetization in the plane of the sample - system lies between a continuous film and an array of independent dots

S-layer on Si

Cr (e-beam)

Plasma etch

Fill Pd/ Fe

Remove mask

Malkinski L, Camley RE, Celinski Z, et al. J Appl Phys 93: 7325-7327, 2003

• S-layer self-assembles on Si wafer• Bring into direct contact with a

photomask and expose to “deep” UV irradiation (ArF, 193 nm @ 200 mJ cm-2).

• S-layer entirely ablated from Si substrate to generate long-range crystalline pattern

• A “natural resist” with excellent (~5 nm) edge resolution?

Pum D, Sleytr UB Trends Biotechnol 17: 8-12 1999

S-layers

3 nm

HSP60

• In nature, chaperonins are ubiquitous and essential subcellular structures responsible for ensuring proteins are correctly folded in the cell

• Composed of 14, 16 or 18 subunits called “heat shock proteins” arranged as two stacked rings.

• The extremophile S.shibatae grows at 85°C at pH 2. Chaperonin is octadecameric with 9 subunits per ring 18 nm tall 17 nm wide.

• Thermostable, of known sequence, and can adopt higher-order structures such as 2-D crystals

McMillan RA, Paavola CD, Howard J, et al. Nat Mater 1: 247-252, 2002

9 nm

HSP60

• Mutant protein assembled into hexagonally packed 2-D arrays ~20 m diameter.

• Added commercial gold QDs• Mutant protein with 3 nm thiol ring

bound and ordered 5 nm particles in pore region; did not bind/ order 10 nm & 15 nm particles

• Mutant protein with 9 nm thiol ring bound and ordered 10 nm particles in pore region….

• Centre-to-centre spacing of 16 nm; edge to edge spacing 6-10 nm

• Also bound & ordered 4.5 nm Cd/Se-ZnS QDs - mottled

• React gold nanoparticles (1.4 nm) with protein subunits prior to self-assembly

• Observed ordered hexagonally spaced inclusions, with density ~9 times greater than single gold nanodot

• Defect tolerant?• Protein intrinsic to design, but

super-stable complex

HSP60

Ferritin

• 24 subunits, 8-9 nm central cavity• Naturally chelates iron (III) as

ferrihydrite• A set of moulds for making

nanometer-scale machine parts with controlled shapes and compositions?

• Synthetic forms created to contain manganese oxide, uranyl oxyhydroxide, iron sulfide, CdS, Ni, Co/Pt

• Co/Pt and Fe-Pt selected as thermally stable at 3-4 nm diameter

Ferritin in magnetic media?

• Superparamagnetic Co-Pt prepared within apoferritin in solution• Self-assembles at an air/water interface giving a well-ordered array

of L-ferritin on Si.

• Anneal at 500-650ºC for 60 min to form ferromagnetic L10 phase and carbonise protein (UV/ ozone treatment decreases sintering)

• Hard disk drives information density restricted by coarse patterning/ coarse grain size and superparamagnetic effect

• Self-assembly of ferritin followed by carbonisation: maximum ~5,000-10,000 Gbits in-2

Ferritin organised on S-layers

50nm

Bacteriorhodopsin

• Found mainly in the halophilic archae – halobacteria

• Exist in a 2D crystalline form in the membrane (“purple membrane”)

• Stable in 5 M (25 %) NaCl, exposure to sunlight in oxygen (years), temperatures ~140ºC when dry, pH values 0-12, enzyme digestion

• Converts light energy into chemical energy

• Simplest photosynthetic machinery – drives an ATP synthetase in the membrane under anaerobic conditions

Bacteriorhodopsin

Bacteriorhodopsin

• Photon absorbed – photochemical switch in the retinal (isomerisation) driving proton pump, proton motive force drives ATP synthase

• Thermal relaxations regenerate ground state.

• Non physiological conditions and intense light – can access branched photocycle and populate the P/Q forms

• P/Q are stable for decades – do not undergo thermal relaxation

BR in holographic memory

BR in holographic memory

Advantages?

Low cost of molecules Self assembly at some stages. No moving partsStorage density? 1x1x2in ~1Tbyte

Limitations?protein purityLaser focusing

Summary

• Proteins have useful self-assembly properties (Viral toolkit, S-layers, HSP60, Ferritin, Bacteriorhodopsin)

• Genetic engineering allows production of entirely new functional sequences

• Protein not necessarily intrinsic to design, but can select for (or evolve) very stable varieties

SyllabusThe molecules of lifeProteins (6 lectures)

backgroundas components in nanodevices biomolecular electronic devices electron transport and photosynthesis as fibrous materials in motion – molecular motors

DNA (3 lectures)background as components in nanodevices: part Ias components in nanodevices: part II

Lipids (1 lecture)background; as components in nanostructures:

artificial cells (liposomes and membrane nanotubes)

Bio-inorganic composites (1 lecture)composites – including butterfly wings, diatoms,

mineralisation

The whole cell Cell mechanotransduction (1 lecture)

bringing together physical, life, and applied sciences; bone cell mechanobiology

 Cell motility (1 lecture)

how cells travel and navigate through 2- and 3 dimensional environments

 Biomaterials (1 lecture)

surface science/ surface chemistry; tissue engineering

 Nanomedicine (1 lecture)

Nanotherapeutics, real and imagined·        Qdots and developmental biology

 Ethical considerations (1 lecture)

risk/benefit analysis focusing on bio-nanotechnology