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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
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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