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Lecture 7 Biomotors

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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Lecture 7 Biomotors

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  1. Lecture 7 Biomotors Linear motors on tracks

  2. Examples of Biomolecular Motors Karplus and Gao, Curr Opin. Struct. Biol (2004) 250-259

  3. Myosin motor pulls on actin filaments Actin and Myosin - Muscle power

  4. Myosin power strokke driven by ATP hydrolysis

  5. Watching individual actin filaments driven by myosin Actin filaments - 8nm in diameter

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

  7. 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

  8. Tubulin - a self-assembling, re-modellable track

  9. Lecture 8 Designed self-assemblywith Biomolecules Polypeptide vs DNA

  10. Self-assembly of polypeptides - fibres and tubes Rajagopal and Schneider Curr Opin. Struct. Biol (2004) 14 p480-6

  11. 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

  12. ‘Amyloid’ fibres - a generic protein/peptide aggregate Peptide Aggregation Nucleus Protofilament Peptide fibril Fibre

  13. Peptide nanotubes - a silver cloud with a peptide lining Reches and Gazit Science (2003) 300, p625

  14. Lecture 8 Designed self-assemblywith Biomolecules Polypeptide vs DNA

  15. Nucleic Acid - the Basics Nucleic acid bases Adenine (A) Guanine (G) Cytosine (C) Thymine (T; R = CH3) Pyrimidines Purines NB – structural similarity

  16. Nucleic Acid - the Basics Nomenclature 2´-deoxyribonucleoside deoxycytidine deoxyadenosine deoxyguanosine thymidine (or deoxythymidine) (deoxyuridine) cytosine deoxyribose base + sugar= nucleoside

  17. Nucleic Acid - the Basics Nomenclature cytosine 2´-deoxyribonucleotide deoxycytidine-5´-monophosphate 5´-dCMP (or just dCMP) deoxyribose base + sugar+ phosphate= nucleotide

  18. Nucleic Acid - the Basics DNA strands Long polymer Base Sugar Phosphate Phosphodiester bond Sugar-phosphate backbone Nucleotide

  19. Nucleic Acid - the Basics Base pairing

  20. 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

  21. 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?

  22. 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

  23. 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.

  24. Nucleic Acid - the Basics Supercoiling Coil Supercoil

  25. Replication

  26. Translation

  27. Nucleic Acid - the Basics Sticky ended ligation Annealing Ligation

  28. Nucleic Acid - the Basics Strand exchange - junctions and branches Holliday Junctions Double Crossover Molecules

  29. Nanostructured Nucleic Acid Materials - Ned Seeman Nature 421 (2003) p427

  30. Tiling with DNA

  31. Tiling with DNA

  32. DNA ‘motors’ - DNA as fuel Seeman

  33. 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

  34. 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)

  35. 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)

  36. Copying DNA - the polymerase chain reaction

  37. Copying DNA - the polymerase chain reaction

  38. Copying DNA - the polymerase chain reaction

  39. 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

  40. DNA detection using nanoparticle assembly Chad Mirkin Thiol terminated ssDNA Sensitivity - femtomol(ar) Selectivity - 100,000 : 1 for point mutations (singlr base pair changes)

  41. DNA detection using nanoparticle assembly Chad Mirkin

  42. Using DNA bar codes to detect proteins Chad Mirkin Science 2003, 301, 1884-1886.

  43. Using DNA bar codes to detect proteins Chad Mirkin Sensitivity 3 aM 30 aM aM = attomolar = 10-18M Science 2003, 301, 1884-1886.

  44. Protein diagnostics using DNA Niemeyer DNA protein conjugates - ImmunoPCR

  45. DNA as a scaffold for something else Biotin Streptavidin interaction - generic molecular adapters

  46. DNA as a scaffold for something else Niemeyer DNA directed immobilisation (DDI) Niemeyer Enzyme locaisation

  47. Protein directed DNA organisation Niemeyer Chains Rings Networks Ionic strength dependent supercoliing

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