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Today ’ s Topic: AFM

Today ’ s Topic: AFM. Experimental Approach via Atomic Force Microscopy Imaging Mode, Force Mode. Example: Measuring strength of Heart Muscle ( Titin ) Strength of a single Covalent Bond Imaging: Correlation Functions. AFM — Force. Force – one place.

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Today ’ s Topic: AFM

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  1. Today’s Topic: AFM • Experimental Approach via Atomic Force Microscopy • Imaging Mode, Force Mode. • Example: Measuring strength of Heart Muscle (Titin) • Strength of a single Covalent Bond • Imaging: Correlation Functions

  2. AFM — Force Force – one place http://cp.literature.agilent.com/litweb/pdf/5990-3293EN.pdf http://www.home.agilent.com/agilent/editorial.jspx?cc=US&lc=eng&ckey=1774141&nid=-33986.0.02&id=1774141

  3. Reversible Unfolding by AFM Pulling on Titin Why does curve look like it does? Why non-linear? Why repeat? Does repeat tell you anything about polymer? 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. Gold Reversible Unfolding of Individual Titin Immunoglobulin Domains by AFM, Science, M. Reif, H. Gaub, 1997

  4. Example of AFM-Force: Muscle & Titin

  5. The Sarcomere: unit of muscle Myosin head binds to actin; rotates upon ATP binding, pulls actin together.

  6. Myosin II moves Actin Notice: ATP induces a conformational change: rotation of lever arm Myosin II acting as a fulcrum, rotating with ATP while driving actin 2 heads of Myosin II; only one head per dimer active. Myosin II spends about 5% of it’s time bound to actin. Myosin II is a non-processive motor, i.e. by itself, it takes 1 step on actin. is processive only because it works in groups which are held together via the thick filament. (Vale & Milligan, Science)

  7. Titin: Human’s Biggest protein Silicon Nitride lever: 10’s pN – several nN’s measureable Each domain IgG Titin: 4.2MDa; Gene (on # 2) = 38,138 aa: Goes from Z-disk to Center; stretchy =I Band Cardiac (N2B &N2BA), Skeletal (N2A), Smooth all have different regions.

  8. Picking up a single protein “needle in a haystack”: usually pick up > 1 protein “Fingerprint” of e.g. (I91)8: by using identical repeats, unfolding forces are nearly identical with peaks equally spaced. (see Fig d) Protein stretched at constant velocity Titin: ≈ 1 um/sec Physiological range Worm-like Chain (WLC) is very good approximation to F vs. x of individual unit (protein, DNA) expansion.

  9. Out1 P N N In1 In2 P Out2 Position sensitive detector (PSD) Useful in AFM, Optical Traps… SIGNAL POSITION ΔX ~ (In1-In2) / (In1 + In2) ΔY ~ (Out1-Out2) /(Out1+Out2) Over a fairly wide range, it’s linear

  10. Force Spectroscopy How Strong is a Covalent Bond? Recall: what did we say it was? About 100-200 kBT Covalent bond to the tip, substrate-- gold or glass-- and within Amylose. We stretched the molecules until one of the covalent bonds in series ruptured. By analyzing the bond rupture, we were able to identify the bond that failed. Within amylose (covalent bonds) was found not to rupture. How Strong is a Covalent Bond? Gaub, Science, 1999 Note: It’s actually the C-Si which breaks!

  11. Figure 2 (A) Force versus extension curve of amylose covalently bound between an AFM tip and a silicon oxide surface. Control: no covalent attachment with amylose. Reversible stretching of amylose (polysaccharides). Not dependent on rate of stretching. Sugar rings switch into a more extended arrangement. With amylose, this results in a characteristic plateau at 275 pN with an extension of 0.5 Å per ring unit (Fig. 2A). Thus, this transition can be used as a molecular strain gauge that can be built into an experiment to report the force that is acting on any point of the molecular bridge. With amylose: 275 pN (low-force) with an extension of 0.5 Å per ring unit = (275pN)(0.05nm) = 13.75 pN-nm = 3kBT 2B: Covalent attachment: sudden ruptures about 2 pN. At the given force-loading rates of 10 nN/s, the histogram peaks at a value of 2.0 ± 0.3 nN. M Grandbois et al. Science 1999;283:1727-1730 Published by AAAS

  12. Figure 3 (A) Histogram of the length gain after the events were measured in the force versus extension curves showing multiple ruptures for amylose, which was covalently attached to the silicon oxide surface and the tip. No EDC or NHS used. Attachment non-specific: lower force. Pop, pop, pop M Grandbois et al. Science 1999;283:1727-1730 Published by AAAS

  13. A Single Covalent bond F = 2.0 nN = 2000pN C-Si: 0.185 nm (estimate) (2000pN)(0.185 nm) = 370 pN-nm 1kBT = 4pN-nm E = 92.5 kBT Sulfur-gold anchor ruptured at 1.4 +/- 0.3 nanonewtons at force-loading rates of 10 nanonewtons/second. ExampleRupture Force Breaking of a covalent bond C-C ≡ 1600 pN Breaking of a non-covalent bond. Biotin/streptavidin ≡ 160 pN (strongest known) Breaking of a weak bond. Hydrogen bond ≡ 1- 4 pN

  14. Which is Covalent Bond that breaks? Largely theoretical argument. Four bonds are unique to the attachment: Si–O, Si–C, C–C, and C–N bonds. The C–O bond is found in the attachment and in the amylose backbone. At first, it was difficult to decide which of these four different bonds was breaking in our experiment. We ruled out the rupture of the Si–O bond because three of these bonds hold in parallel at the surface. As a first approximation, we correlated the strength of a covalent bond with the ratio of the dissociation energy and the bond length. Considering the enthalpy for dissociation and the bond length (20), we decided that the Si–C bond was the most likely candidate for rupture in our experiment. How Strong is a Covalent Bond? Gaub, Science, 1999

  15. AFM Images Bacteria DNA molecules Mosquito eye http://www.afmhelp.com/index.php?option=com_content&view=article&id=51&Itemid=57

  16. Convolution of tip and sample size Tobacco Mosaic Virus (TMV) In truth, diameter of 180 Å. Due to finite tip size, w~ 350 A http://webserv.jcu.edu/chemistry/faculty/waner/research/AFM/tipconv.htm

  17. If tip size is large, have to worry about distortions. Convolution What will image look like? Typically, probe radius varies from 5 to 20 nm http://webserv.jcu.edu/chemistry/faculty/waner/research/AFM/tipconv.htm

  18. Correlation functions <f(t) * g(t-t)> Cross-correlation Can also do auto-correlation: (as in WLC) Cross- correlation What if red curve is like a delta function (really narrow)? Reproduce blue box What does cross-correlation look like? http://www.scholarpedia.org/article/1/f_noise

  19. 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.

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