AnnouncementsMid-Term Test next Tuesday in class! (Oct. 7th, 9:30-11am, Rm 136LLP)

Will cover all of classes Lec 1-10 plus (qualitatively) on Lec 11– Lec 11: The difference between Hook’s Law, FJC, WLC;

Prions (what they are, why unique, why Nobel Prize)(Today is Lec 12)

Go over:•Lecture notes (on Web).•Make sure you can do all problems on Homework.•Reading: Chpt 1 of Berg; Scientific American Article on Molecular Biol. Of Cell.

The exam: Some qualitative questions (Explain the structure of DNA; explain why the Central Dogma of Molecular Biology has been modified recently and explain why RNA has played a role in this.)Know how many kBT’s are in ATP and how many pN-nm (and why there is a range). Some quantitative questions: G, H, S. Boltzmann Distribution.

Questions related from Last Time

Simple, but excellent Videos about Prions

http://www.youtube.com/watch?v=tfv3xAw0XOE

Simple, but excellent Videos about Prions

http://www.youtube.com/watch?v=pqhpVpafjmk

Two Models of DNA (simple) Freely Jointed Chain (FJC)

& (more complicated) Worm-like Chain (WLC)

Idealized FJC:

Realistic Chain:

FJC: Head in one direction for length b, then turn in any direction for length b.

[b= Kuhn length = ½ P, where P= Persistence

Length]

WLC: Have a correlation length

FJC: Completely straight, unstretchable. No thermal fluctuations away from straight line are allowed

The polymer can only disorder at the joints between segments

FJC: Can think of DNA as a random walk in 3-D.

Today’s Topic: AFM

• Experimental Approach via Atomic Force Microscopy• AFM: Can see Nanometer & Angstrom scale changes!• How you can see this: Hook’s Law• Imaging Mode, Force Mode.• Force Mode: Worm Like Chain model of Protein

Folding (as well as DNA)

http://cp.literature.agilent.com/litweb/pdf/5990-3293EN.pdf

AFM—Imaging

http://www.home.agilent.com/agilent/editorial.jspx?cc=US&lc=eng&ckey=1774141&nid=-33986.0.02&id=1774141

Ionic repulsion,bends tip

Imaging – ScanMuller,

Biochemistry, 2008

Measure (z-axis) distance,Or

Constant force (by altering distance with feedback)

Measuring forcesThe force is not measured directly, but calculated by measuring the deflection of the lever, and knowing the stiffness of the cantilever. Hook’s law gives F = -kz, where F is the force, k is the stiffness of the lever, (in Newtons/meter) and z is the distance the lever is bent.

http://cp.literature.agilent.com/litweb/pdf/5990-3293EN.pdf

Most AFM probes are made from silicon and/or silicon nitride (Si3N4) wafers using semiconductor-based etching processes.

Hook’s Law and AFM

http://www.home.agilent.com/agilent/editorial.jspx?cc=US&lc=eng&ckey=1774141&nid=-33986.0.02&id=1774141

Ionic repulsion,bends tip

Imaging—ScanForce– one place

Can measure Angstrom resolution – Nobel Prize, 1986

What determines how accurately you can measure?

If photodetector was on the moon, could you see infinitely small changes?

Brownian Noise & Equipartition Theorem

Each degree of freedom in Energy goes as x2 or v2 : has ½ kBT of energy.

A gas molecule at temperature T: Kinetic Energy: ½ mv2 : Has ½ kBT of energy

½ mv2 = ½ kT : v2 = kBT/m v = √kT/m : ~ 1000 mph!

If you have a spring at finite temperature:

Examples?

½ kx2 = ½ kBT

AFM CantileverHow small of a motion can you measure?

How to determine?

Bend a cantilever (in z-direction): ½ kz2

½ kBT = ½ kz2 (z2 is the mean square deflection of the cantilever caused by thermal vibrations)

What is k?Stiffer material?h, w, L go up or down?

z2 = kBT/k = 0.64Å/√k at 22˚C (where k is in N/m)

k between 0.001 to 100 N/m (Huge range! Very useful for measuring large ∆z, F: 1 pN - nN) Say, typical: 1 N/m = 1nN/nm: 1 nN causes deviation of 1 nm

1 nN usually really large0.01 N/m = 10 pN/nm: 1 pN would cause a deviation of 1 nm)

Can measure an Angstrom or less!!

k = 0.25Ewh3/L3, where E = modulus of Elasticity (how stiff the material is).

http://cp.literature.agilent.com/litweb/pdf/5990-3293EN.pdf

AFM — Force

http://www.home.agilent.com/agilent/editorial.jspx?cc=US&lc=eng&ckey=1774141&nid=-33986.0.02&id=1774141

Force – one place

Reversible Unfolding of Individual Titin Immunoglobulin Domains by AFM, Science, M. Reif, H. Gaub, 1997

Reversible Unfolding by AFM Pulling on Titin

Gold

Simple model: Upon reaching a certain force (peaks, e.g. 1), the abrupt unfolding of a (Titin) domain lengthens the polypeptide by 28 to 29 nm and reduces the force (troughs) to that of the value predicted by the force extension curve of the enlarged polypeptide (2). Start on next domain. As it’s pulling, polymer behaves like WLC.

Why does curve look like it does?Why non-linear?Why repeat?Does repeat tell you anything about polymer?

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.