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Lecture 7 Biomotors

This lecture explores biomolecular motors like myosin and kinesin, which power cellular movements by interacting with actin and microtubules, respectively. Myosin, fueled by ATP hydrolysis, enables muscle contraction by pulling on actin filaments. Kinesin, as a motor protein, travels along microtubules, a vital process for intracellular transport. The presentation also covers foundational knowledge of nucleic acids, their structure, energetics, and the innovative use of DNA as nanoscale motors and materials, highlighting the significance of biomolecular self-assembly.

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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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