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Lecture 7 Biomotors. Linear motors on tracks. Examples of Biomolecular Motors. Karplus and Gao, Curr Opin. Struct. Biol (2004) 250-259. Myosin motor pulls on actin filaments. Actin and Myosin - Muscle power. Myosin power strokke driven by ATP hydrolysis.

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slide1

Lecture 7 Biomotors

Linear motors on tracks

slide2

Examples of Biomolecular Motors

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

slide4

Myosin motor pulls on actin filaments

Actin and Myosin

- Muscle power

slide7

Kinesin

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

slide8

1 monomer

Watching kinesin walk.

  • The motor protein kinesin walks along microtubules, one tubulin subunit at a time
  • using an optical trap, one can follow its steps
slide11

Self-assembly of polypeptides - fibres and tubes

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

slide12

Self-assembly of polypeptide secondary structures

MacPhee and Woolfson Curr Opin. Solid-state and Materials Science

(2004) 8 p141-149

b-sheet ‘amyloid’-type

Protein fibrils

a-helix coiled-coil-type

protein fibrils

slide13

‘Amyloid’ fibres - a generic protein/peptide aggregate

Peptide Aggregation Nucleus Protofilament Peptide fibril Fibre

slide14

Peptide nanotubes - a silver cloud with a peptide lining

Reches and Gazit Science (2003) 300, p625

nucleic acid bases

Nucleic Acid - the Basics

Nucleic acid bases

Adenine (A)

Guanine (G)

Cytosine (C)

Thymine (T; R = CH3)

Pyrimidines

Purines

NB – structural similarity

nomenclature

Nucleic Acid - the Basics

Nomenclature

2´-deoxyribonucleoside

deoxycytidine

deoxyadenosine

deoxyguanosine

thymidine

(or deoxythymidine)

(deoxyuridine)

cytosine

deoxyribose

base + sugar= nucleoside

nomenclature1

Nucleic Acid - the Basics

Nomenclature

cytosine

2´-deoxyribonucleotide

deoxycytidine-5´-monophosphate

5´-dCMP (or just dCMP)

deoxyribose

base + sugar+ phosphate= nucleotide

dna strands

Nucleic Acid - the Basics

DNA strands

Long polymer

Base

Sugar

Phosphate

Phosphodiester bond

Sugar-phosphate backbone

Nucleotide

canonical w c structure

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
dna structure variations

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?
dna energetics

Nucleic Acid - the Basics

DNA energetics
  • DNA can be reversibly denatured ("melting")
    • Cooperative transition from helix  random coil; the change in absorbance at l=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
dna energetics1

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.
slide29

Nucleic Acid - the Basics

Sticky ended ligation

Annealing

Ligation

slide30

Nucleic Acid - the Basics

Strand exchange - junctions and branches

Holliday

Junctions

Double

Crossover

Molecules

slide35

DNA ‘motors’ - DNA as fuel

Tuberfield

Nature 406 (2000)

P605-8

Seeman

‘Biped’ Nanoletters 4 (2004) p 1203-7

Proof??

Video

Liao and Seeman

Science 306 (2004) 2072-2074

Links to DNA synthesis

slide37

Alternative DNA structures - G-quadruplexes

Assembly of a nanoscale quadruple helix

Balasubramanian and co-workers

J. Am. Chem. Soc. 126, 5944-5945 (2004)

J. Am. Chem. Soc. 125, 11009-11016 (2004)

slide38

DNA ‘motors’ - Protons as fuel

Proton driven single molecule DNA motor

OH-

H2O

i-motif

H+

H2O

Balasubramanian and co-workers

Angew. Chem. Intl. Ed., 42, 5734-5736 (2003)

slide42

Attaching things to DNA

Biotin Streptavidin interaction - generic molecular adapters

Thiols - Nanoparticles

Fluorohores - for sensitive detection

Proteins - protein/DNA recognition

Proteins - semi-synthetic conjugation

Metal - metallisation for conductors

slide43

DNA detection using nanoparticle assembly

Chad Mirkin Thiol terminated ssDNA

Sensitivity - femtomol(ar)

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

slide45

Using DNA bar codes to detect proteins

Chad Mirkin

Science 2003, 301, 1884-1886.

slide46

Using DNA bar codes to detect proteins

Chad Mirkin

Sensitivity

3 aM

30 aM

aM = attomolar = 10-18M

Science 2003, 301, 1884-1886.

slide47

Protein diagnostics using DNA

Niemeyer DNA protein conjugates - ImmunoPCR

slide48

DNA as a scaffold for something else

Biotin Streptavidin interaction - generic molecular adapters

slide49

DNA as a scaffold for something else

Niemeyer DNA directed immobilisation (DDI)

Niemeyer Enzyme locaisation

slide50

Protein directed DNA organisation

Niemeyer

Chains Rings Networks

Ionic strength dependent supercoliing

slide51

DNA directed Protein organisation

Niemeyer Enzyme localisation

ChemBioChem (2003) 2, p242-245

slide52

DNA (and protein) metallisation

Braun, Finkelstein and others

Yan et al Science (2003) 301 p1882

slide53

DNA (and protein) metallisation

Braun, Finkelstein and others

Yan et al Science (2003) 301 p1882