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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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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’-type Protein fibrils -helix coiled-coil-type protein fibrils Self-assembly of polypeptide secondary structures
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Peptide Aggregation NucleusProtofilamentPeptide 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 Adenine (A)Guanine (G) Purines Cytosine (C)Thymine (T; R = CH 3 ) Pyrimidines NB – structural similarity Nucleic Acid - the Basics
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Nomenclature base + sugar = nucleoside deoxyribose cytosine 2´-deoxyribonucleoside deoxycytidine deoxyadenosine deoxyguanosine thymidine (or deoxythymidine) (deoxyuridine) Nucleic Acid - the Basics
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Nomenclature deoxyribose cytosine 2´-deoxyribonucleotide deoxycytidine-5´-monophosphate 5´-dCMP (or just dCMP) 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 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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Replication
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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 Crossover Molecules
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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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DNA ‘motors’ - DNA as fuel Seeman
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DNA ‘motors’ - DNA as fuel Seeman ‘Biped’ Nanoletters 4 (2004) p 1203-7 Proof?? Tuberfield Nature 406 (2000) P605-8 Video Liao and Seeman Science 306 (2004) 2072-2074 Links to DNA synthesis
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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) Alternative DNA structures - G-quadruplexes
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OH - H+H+ H2OH2O H2OH2O 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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Attaching things to DNA 1.Biotin Streptavidin interaction - generic molecular adapters 2.Thiols - Nanoparticles 3.Fluorohores - for sensitive detection 4.Proteins - protein/DNA recognition 5.Proteins - semi-synthetic conjugation 6.Metal - metallisation for conductors
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DNA detection using nanoparticle assembly Chad MirkinThiol 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 -18 M
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NiemeyerDNA 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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NiemeyerDNA directed immobilisation (DDI) DNA as a scaffold for something else NiemeyerEnzyme locaisation
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Niemeyer Protein directed DNA organisation ChainsRingsNetworks Ionic strength dependent supercoliing
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DNA directed Protein organisation NiemeyerEnzyme localisation ChemBioChem (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
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