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(530190) Methods in Single Molecule Biophysics, (3op) , Fall 2011
•Lectures: Anders Wallin ([email protected]) •Exercises: Kalle Hanhijärvi ([email protected])
• http://electronics.physics.helsinki.fi
•7 Lectures, 2h each :
•Tuesday 14-16 in D116 (1+2.11 8.11 15.11 22.11 29.11 6.12(no lecture!) 13.12)
•Exercises: ~6 weeks, Wednesday 12-13 in D116
9.11 16.11 23.11 30.11 7.12 14.12
•Presentation if one single-molecule paper by each student on 13.12 •Exam: sometime 15-20.12
•Grade: Exercises 30% (be present!), Presentation, Exam 70%
Content (preliminary) • 1 Introduction
– Introduction and motivation – Applications (what biological
questions are there?) – Methods (how do we try to
answer the questions?) • 2 Methods
– Optical Tweezers – Magnetic tweezers – AFM – Brownian Dynamics (Langevin
eq. Fokker-Planck?) – Physics of detection
• 3 Polymer/chain models – Polymers (DNA) – Freely jointed chain – Worm-like chain – DNA/RNA Hairpin models?
• 4 Motors – Molecular motors (Kinesin, Myosin etc) – Models (Brownian ratchet, etc)
• 5 Imaging & Fluorescence – Single molecule fluorescence – Diffraction & resolution – Superresolution methods
• 6 Applications & Examples – E.g. Motility, DNA-replication&repair – Recent papers
Material • Lecture notes + exercises
– Will be available on the homepage • Books
– Philip Nelson: Biological Physics: Energy, Information, Life, W. H. Freeman (July 18, 2003)
– Jonathon Howard: Mechanics of Motor Proteins and the Cytoskeleton Sinauer Associates Incorporated; New Ed edition (February 16, 2001)
– Knight: Single Molecule Biology
• Review papers • Video lectures
1 Why Single molecules? 2 Some biological problems 3 Some Methods
Nanoscience?
Bacterial (prokaryotic) cells:
smaller, highly adaptive fast growing, mobile, simple
sensory system
Eukaryotic cells: larger, grow slower in well-defined environment, organelles, cell-cell signaling, differentiation, development, multicellular organisms
8
The cell: a collection of molecular machines
[Goodsell]
The Central Dogma
The central dogma states that information in nucleic acid (DNA in cells) can be perpetuated (replication) or transferred (transcription), but the transfer of information form into protein (translation) is irreversible.
Why single molecules?
• no need for sample synchronization
• detection of intermediates
• single molecule measurements avoid ensemble averaging
• full statistical description can be achieved
• When does averaging work? • Do we need to observe a single molecule ?
11
Why single molecule biology?
Does nature look like this?
... Or this?
Why Single Molecule Biophysics ?
• Ensemble measurements give: Averages over large populations (+errors for these averages)
• Single Molecule measurements give:Distributions ! • Rare events!
Adelman et. al., PNAS, 2002, Vol 99 no 21
Single Molecule Biology • DNA
– What kind of a polymer is DNA ? – How do DNA-enzymes work ? – DNA-compaction
• The folding problem – Proteins – RNA
• Molecular motors/machines – How do they work? – How is chemical energy converted to mechanical
work? – Can we engineer motors?
DNA properties
DNA properties
RNA Folding
molecular motors • sizes ~ 10 nm (head) ~ 100
nm (overall) • forces ~ 5 pN • speeds ~ 10 μm/s • stiffness ~ 1 pN/nm • diffusion coeff. ~ 10 mm2/s
(head diffuses 10 nm in 10 ms in free solution)
• cycle time ~ 20 ms (ca 50 steps/s)
• hydrolysis energy ~ 20 kBT = 80 pN nm Hoenger et al., J Mol Biol, 297, 1087-103
Examples of Molecular Motors
Myosin is present in human skeletal muscle Myosin steps along an Actin track Kinesin transports various cargos (vesicles) inside the cell Kinesin steps along a Mircotubule track
Videos of optical tweezers
• Stretching DNA in optical tweezers: http://www.youtube.com/watch?v=HsuwMnMIEyc
• Lambda exonuclease, a simple molecular motor: http://www.youtube.com/watch?v=ddPdxk6-1k0
