Announcements Lec 10

1. AnnouncementsLec 10 Homework #5 Assigned includes Reading, Chpt 3 ECB Most students like in-class assignments (good!) Quiz today--ATPase

2. Quiz #4 beta (1) F1 ATPase has three ______________ subunits and three _____________ subunits arranged in a ringlike structure. In the center there is a _______________ shaft. (2) There are three ATP binding sites in the F1 motor, what are the nucleotide states of each site? 1- 2- 3- (3) In Noji et al. Nature 1997, it was shown that F1 has 120 degree steps. However, in the review article it is mentioned that these steps are further broken into one _________ and one _________ degree sub-steps. (4) The whole ATP synthase does / does not rotate. alpha gamma ATP ADP+Pi empty 90 30 2/13/08

3. Accuracy vs. Resolution Accuracy: Unlimited: limited by # sqrt(photons) Resolution:Can’t get better than l/2N.A. A single spot will be smeared out, no matter how small the spot is, because of the wavelength of light to ~ l/2N.A. (shape = Point Spread Function, PSF). Or can you? We can in-fact, achieve sub-diffraction limited resolution by several ways.

5. Electron Microscope Images (Usually, but not always, requires staining with metals to get sufficient contrast) [For Transmission E.M. need to make very thin sections.]

6. Scanning E.M. (Electrons scattered from surface) Specimen is coated with heavy metals. Raster beam across specimen. Good depth of focus. Amount of electrons scattered depends on angle of surface – gives highlights, shadows, 3-d surfaces. D.I.C. (Phase)

9. sin q = nl/d d ~ 2 Å ; l ~ 1-2 Å

10. a-helix 20 Å Measuring atomic distances X-ray crystallography Another technique: NMR Scattering electron density Atomic Myoglobin crystal Stryer, pg. 65 backbone

11. Optical Scattering Analog

13. Crystal of protein must tell you something about structure in water. X-ray Crystallography: must crystallize to get reasonable diffraction. (With partially oriented samples, like original DNA, can get some information– see next slide) NMR– in solution. NMR is better, except very limited what range it can do usually <25k Blue = x-ray crystallography; red = NMR Structure of pltocyanin (protein in photosynthesis) Often-times (though not always), get good agreement!

15. FIONA Fluorescence Imaging with One Nanometer Accuracy (1.5 nm, 1-500 msec)

16. Single Molecule Studies of Molecular Motors In vitro & in vivo How are biomolecules moved around within a cell? “Kinesin can carry a packet of neurotransmitter from your spine to the tip of your finger in about two days — a journey that would take a thousand years if left to simple diffusion.” (Molloy and Schmitz, Nature, 2005)

17. Biomolecular Motors: Intra- & Extra-Cellular Motion ATP-binding heads Actin, mtubules Actin, mtubules ATP  mechanical work Nature Reviews Cargo binding Kinesin Myosin Dynein  Motor Microtubule actinMicrotubule polymer (+) direction (- direction) • nm scale • Move along tracks w ATP • intracellular directional movement • cell shape & extracellular movement

18. Myosin V (Kinesin): Hand-over-hand or Inchworm? Kinesin Myosin V Kinesin: 0, 16.6 nm or 8.3, 8.3 nm Myosin V: 0, 74 nm or 37, 37 nm

19. Class evaluation • What was the most interesting thing you learned in class today? • 2. What are you confused about? • 3. Related to today’s subject, what would you like to know more about? • 4. Any helpful comments. Answer, and turn in at the end of class.