AnnouncementsLec 10

Quiz today--ATPase

Homework #5 Assigned

includes Reading, Chpt 3 ECB

Most students like in-class assignments (good!)

Quiz #4

(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.

betaalpha

ATPADP+Piempty

3090

gamma

2/13/08

Resolution: Can’t get better than /2N.A.

Accuracy vs. Resolution

A single spot will be smeared out, no matter how small the spot is, because of the wavelength of light

to ~ /2N.A.

(shape = Point Spread Function, PSF).

Or can you? We can in-fact, achieve sub-diffraction limited resolution by several ways.

Accuracy: Unlimited: limited by # sqrt(photons)

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.]

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)

sin = n/d

d ~ 2 Å ; ~ 1-2 Å

Measuring atomic distances X-ray crystallography

Another technique: NMR

Myoglobin crystal

Stryer, pg. 65

20 Å

-helix

Atomic

Scattering electron density

backbone

Optical Scattering Analog

Crystal of protein must tell you something about structure in water.

Structure of pltocyanin (protein in photosynthesis)

Blue = x-ray crystallography; red = NMR

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

Often-times (though not always), get good agreement!

Fluorescence Imaging with One Nanometer Accuracy

(1.5 nm, 1-500 msec)

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)

Single Molecule Studies of Molecular Motors

In vitro & in vivo

Biomolecular Motors: Intra- & Extra-Cellular Motion

Act

in,

tub

ule

s

ATP mechanical work

Nat

ure

Rev

iew

s

Microtubule actin Microtubule polymer

(+) direction (- direction)

Kinesin Myosin Dynein Motor

ATP-bindingheads

Cargo binding

Act

in,

tub

ule

s

• nm scale

• Move along tracks w ATP

• intracellular directional movement

• cell shape & extracellular movement

Kinesin: 0, 16.6 nm or 8.3, 8.3 nm

Myosin V: 0, 74 nm or 37, 37 nm

Kinesin

Myosin V

Myosin V (Kinesin): Hand-over-hand or Inchworm?

Class evaluation

1. 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.