Lecture 7 Biomotors

Linear motors on tracks

Examples of Biomolecular Motors

Karplus and Gao, Curr Opin. Struct. Biol (2004) 250-259

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Actin and Myosin

- Muscle power

Myosin motor pulls on actin filaments

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Myosin power strokke driven by ATP hydrolysis

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Watching individual actin filaments driven by myosin

Actin filaments - 8nm in diameter

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http://www.hybrid.iis.u-tokyo.ac.jp/research.htm

Kinesin

• The motor protein kinesin walks along microtubules, one tubulin subunit at a time

• using an optical trap, one can follow its steps

1 monomer

Watching kinesin walk.

Tubulin - a self-assembling, re-modellable track

Lecture 8 Designed self-assembly

with Biomolecules

Polypeptide vs DNA

Rajagopal and Schneider Curr Opin. Struct. Biol (2004) 14 p480-6

Self-assembly of polypeptides - fibres and tubes

MacPhee and Woolfson Curr Opin. Solid-state and Materials Science (2004) 8 p141-149

-sheet ‘amyloid’-typeProtein fibrils

-helix coiled-coil-typeprotein fibrils

Self-assembly of polypeptide secondary structures

Peptide Aggregation Nucleus Protofilament Peptide fibril Fibre

‘Amyloid’ fibres - a generic protein/peptide aggregate

Peptide nanotubes - a silver cloud with a peptide lining

Reches and Gazit Science (2003) 300, p625

Lecture 8 Designed self-assembly

with Biomolecules

Polypeptide vs DNA

Nucleic acid bases

N

N N

N

NH2

H

N

N N

N

H2N

O

H

H

Adenine (A) Guanine (G)

Purines

N

NO

NH2

H

N

N

O

O

R

H

H

Cytosine (C) Thymine (T; R = CH3)

Pyrimidines

NB – structural similarity

Nucleic Acid - the Basics

Nomenclature

N

NO

NH2

H

base + sugar = nucleoside

deoxyribose

cytosine2´-deoxyribonucleosidedeoxycytidinedeoxyadenosinedeoxyguanosinethymidine(or deoxythymidine)(deoxyuridine)

H

OH

OO CH2

H H

H

H

5´

4´

3´ 2´

1´

H

Nucleic Acid - the Basics

Nomenclature

N

NO

NH2

H

deoxyribose

cytosine2´-deoxyribonucleotidedeoxycytidine-5´-monophosphate5´-dCMP (or just dCMP)

H

OH

OO CH2

H H

H

H

5´

4´

3´ 2´

1´

H

O

O

O

P

–

–

base + sugar + phosphate = nucleotide

Nucleic Acid - the Basics

DNA strands

Long polymer

Base

Sugar

Phosphate

Phosphodiester bond

Sugar-phosphate backbone

Nucleotide

Nucleic Acid - the Basics

Base pairing

N

NN

NN H

H

H

CH 3O

O

NN

NN

NO

N

N

H

H

H

H

H

N

O

NN

A

G

T

C

Nucleic Acid - the Basics

Canonical W-C structure

• B-DNA• Physiologically significant

conformation• Right handed helix• Diameter is ~20 Å• Base tilt to helix axis ~6°• Helical twist per base pair

~34°• 3.4 Å /bp• 10.5 bp /turn

Nucleic Acid - the Basics

DNA structure - variations

• Bases are not flat, but are twisted with respect to each other

• The rotation from one bp to the next is also variable (27-40°)

• Structure of DNA is therefore sequence dependent – identifiable binding sites for regulatory proteins?

Nucleic Acid - the Basics

DNA energetics

• DNA can be reversibly denatured ("melting")– Cooperative transition from helix random coil; the change in

absorbance at =260 nm can be used to monitor this transition. The absorbance (A260) increases when the DNA melts

– Tm (the midpoint) increases with G + C content– Tm increases with increased salt concentration

• Base pairing– Watson-Crick H-bonding is only a minor contribution to stability but

is essential for specificity

• Repulsion between phosphates is minimized by maximizing P -P distance and by interactions with cations

Nucleic Acid - the Basics

DNA energetics

• Base stacking is the major contribution to helix stability.• Planar aromatic bases overlap geometrically and electronically.• Energy gain by base stacking is due to:

– Hydrophobic effect, water is excluded from the central part of the helix, but still fills the grooves. This is a minor contribution to the energy.

– Direct interaction between the nucleotide bases. This is the major favourable contribution to the energetics of DNA folding.

Nucleic Acid - the Basics

Supercoiling

Supercoil

Coil

Nucleic Acid - the Basics

A T AG C A GG T C CT T A CG

T A TC G T CC A G GA A T GC

DNA double helix DNA single strands Two DNA double helices

Replication

DNA double helix DNA single strands DNA–RNA hybrid

Messenger RNA

Protein

Ribosome

Translation

Nucleic Acid - the Basics

Sticky ended ligation

Annealing

Ligation

Nucleic Acid - the Basics

Strand exchange - junctions and branches

Holliday Junctions

Double CrossoverMolecules

Nanostructured Nucleic Acid Materials - Ned Seeman

Nature 421 (2003) p427

Tiling with DNA

Tiling with DNA

DNA ‘motors’ - DNA as fuel

Seeman

DNA ‘motors’ - DNA as fuel

Seeman

‘Biped’ Nanoletters 4 (2004) p 1203-7

Proof??

TuberfieldNature 406 (2000)P605-8

Video

Liao and SeemanScience 306 (2004) 2072-2074

Links to DNA synthesis

Assembly of a nanoscale quadruple helix

Balasubramanian and co-workersJ. Am. Chem. Soc. 126, 5944-5945 (2004)J. Am. Chem. Soc. 125, 11009-11016 (2004)

Alternative DNA structures - G-quadruplexes

OH-

H+

H2O

H2O

i-motif

Proton driven single molecule DNA motor

Balasubramanian and co-workers Angew. Chem. Intl. Ed., 42, 5734-5736 (2003)

DNA ‘motors’ - Protons as fuel

Copying DNA - the polymerase chain reaction

Copying DNA - the polymerase chain reaction

Copying DNA - the polymerase chain reaction

Attaching things to DNA

1. Biotin Streptavidin interaction - generic molecular adapters2. Thiols - Nanoparticles3. Fluorohores - for sensitive detection4. Proteins - protein/DNA recognition5. Proteins - semi-synthetic conjugation6. Metal - metallisation for conductors

DNA detection using nanoparticle assembly

Chad Mirkin Thiol terminated ssDNA

Sensitivity - femtomol(ar)Selectivity - 100,000 : 1 for point mutations (singlr base pair changes)

Chad Mirkin

DNA detection using nanoparticle assembly

Chad Mirkin

Using DNA bar codes to detect proteins

Science 2003, 301, 1884-1886.

Chad Mirkin

Using DNA bar codes to detect proteins

Science 2003, 301, 1884-1886.

3 aM

30 aM

Sensitivity

aM = attomolar = 10-18M

Niemeyer DNA protein conjugates - ImmunoPCR

Protein diagnostics using DNA

DNA as a scaffold for something else

Biotin Streptavidin interaction - generic molecular adapters

Niemeyer DNA directed immobilisation (DDI)

DNA as a scaffold for something else

Niemeyer Enzyme locaisation

Niemeyer

Protein directed DNA organisation

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Chains Rings Networks

Ionic strength dependent supercoliing

DNA directed Protein organisation

Niemeyer Enzyme localisationChemBioChem (2003) 2, p242-245

DNA (and protein) metallisation

Braun, Finkelstein and others

Yan et al Science (2003) 301 p1882

DNA (and protein) metallisation

Braun, Finkelstein and others

Yan et al Science (2003) 301 p1882