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Lecture 7 Biomotors
Linear motors on tracks
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Examples of Biomolecular Motors
Karplus and Gao, Curr Opin. Struct. Biol (2004) 250-259
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QuickTime™ and aCinepak decompressor
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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
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• 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.
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Tubulin - a self-assembling, re-modellable track
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Lecture 8 Designed self-assembly
with Biomolecules
Polypeptide vs DNA
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Rajagopal and Schneider Curr Opin. Struct. Biol (2004) 14 p480-6
Self-assembly of polypeptides - fibres and tubes
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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
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Peptide Aggregation Nucleus Protofilament Peptide fibril Fibre
‘Amyloid’ fibres - a generic protein/peptide aggregate
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Peptide nanotubes - a silver cloud with a peptide lining
Reches and Gazit Science (2003) 300, p625
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Lecture 8 Designed self-assembly
with Biomolecules
Polypeptide vs DNA
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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
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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
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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
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DNA strands
Long polymer
Base
Sugar
Phosphate
Phosphodiester bond
Sugar-phosphate backbone
Nucleotide
Nucleic Acid - the Basics
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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
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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
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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
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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
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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
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Supercoiling
Supercoil
Coil
Nucleic Acid - the Basics
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A T AG C A GG T C CT T A CG
T A TC G T CC A G GA A T GC
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DNA double helix DNA single strands Two DNA double helices
Replication
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DNA double helix DNA single strands DNA–RNA hybrid
Messenger RNA
Protein
Ribosome
Translation
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Nucleic Acid - the Basics
Sticky ended ligation
Annealing
Ligation
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Nucleic Acid - the Basics
Strand exchange - junctions and branches
Holliday Junctions
Double CrossoverMolecules
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Nanostructured Nucleic Acid Materials - Ned Seeman
Nature 421 (2003) p427
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Tiling with DNA
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Tiling with DNA
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DNA ‘motors’ - DNA as fuel
Seeman
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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
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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
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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
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Copying DNA - the polymerase chain reaction
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Copying DNA - the polymerase chain reaction
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Copying DNA - the polymerase chain reaction
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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
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DNA detection using nanoparticle assembly
Chad Mirkin Thiol terminated ssDNA
Sensitivity - femtomol(ar)Selectivity - 100,000 : 1 for point mutations (singlr base pair changes)
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Chad Mirkin
DNA detection using nanoparticle assembly
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Chad Mirkin
Using DNA bar codes to detect proteins
Science 2003, 301, 1884-1886.
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Chad Mirkin
Using DNA bar codes to detect proteins
Science 2003, 301, 1884-1886.
3 aM
30 aM
Sensitivity
aM = attomolar = 10-18M
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Niemeyer DNA protein conjugates - ImmunoPCR
Protein diagnostics using DNA
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DNA as a scaffold for something else
Biotin Streptavidin interaction - generic molecular adapters
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Niemeyer DNA directed immobilisation (DDI)
DNA as a scaffold for something else
Niemeyer Enzyme locaisation
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Niemeyer
Protein directed DNA organisation
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Chains Rings Networks
Ionic strength dependent supercoliing
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DNA directed Protein organisation
Niemeyer Enzyme localisationChemBioChem (2003) 2, p242-245
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DNA (and protein) metallisation
Braun, Finkelstein and others
Yan et al Science (2003) 301 p1882
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DNA (and protein) metallisation
Braun, Finkelstein and others
Yan et al Science (2003) 301 p1882