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Name: Krishnashish Bose Entry no.: 2011PHS 7179

TO DETERMINE MODULUS OF ELASTICITY OF DISEASE-RELATED AMYLOIDS. Name: Krishnashish Bose Entry no.: 2011PHS 7179 Project Supervisors: Prof. B.R.Mehta & Dr. Bishwajit Kundu AFM operator & Technical supervisor: Dr. Deepak Varandani. What are Amyloids?

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Name: Krishnashish Bose Entry no.: 2011PHS 7179

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  1. TO DETERMINE MODULUS OF ELASTICITY OF DISEASE-RELATED AMYLOIDS Name: Krishnashish Bose Entry no.: 2011PHS 7179 Project Supervisors: Prof. B.R.Mehta & Dr. BishwajitKundu AFM operator & Technical supervisor: Dr. Deepak Varandani

  2. What are Amyloids? Amyloids are basically protein nanofibers formed as a result of protein misfolding and ordered aggregation. Parallel chains of long peptide sequences can stack together to form beta sheets which then attach with other beta sheets at the open ends so as to form a closed stable cylindrical structure. The length of the cylinder can vary from 0.1 μm to 0.1 mm. The inner radius could be around 3-10 nm and outer diameter 6-13 nm. Each of these cylinders could bundle together in an alpha helix pattern to form larger fibrils. Isn’t it strange how a protein identifies its own type out of the thousands of other kinds of proteins surrounding it inside the cell !! Not only does it bind to its own type but also in a specific pattern. What if a few of them do not follow a specific pattern? Thank God it’s just a very few, otherwise we would not exist at all. But as usual, there are two sides of every coin. It is observed that some ordered aggregates of misfolded proteins (Amyloids) are not only unique in its morphology but also functionality.

  3. Why do we need to know about nanomechanical properties of amyloids? • It can help us not only to manufacture protein-based nano-devices & structures but also understand some inexplicable cellular/ molecular mechanisms. We can solve the puzzle whether Amyloids are the cause for a disease or its symptom. • The mechanical properties of amyloid fibrils could interfere with cytoskeletal dynamics. They are found to be stiffer than cytoskeleton components such as actin filaments (1.8 GPa), microtubules (600 MPa) or intermediate filaments (6.4 MPa). • Amyloids also play a role as functional nanostructures. Recently, over 30 human peptide hormones were found that are stored in amyloid-type granules for long-term use. This could offer a solution to the short-term delivery problems of drugs by using amyloid as a controlled drug delivery nanodevice. • Due to the self-assembly property of amyloid fibrils, these nanostructures are also interesting as templates to create filaments of other materials that do not have this capability. This can be used to manufacture Nanowires. • Some amyloid fibrils can also bundle together to act as optical waveguides. They can be used in nanoelectronics and nanophotonics.

  4. Prion Nanofibers spotted Nanofibers (possibly)

  5. Nanofibers Nanofibers

  6. Results: Mean Diameter of Prionfibre = (23.735 + 32.112 + 36.328)/3 nm = 30.725 nm Conclusion: The obtained diameter is too big for a single protein nanofiber which is about 10-15 nm. It seems that 2-3 single fibers have bundled together.

  7. Poly Q nanofibers

  8. Results: Mean Diameter of Poly Q fibre 1 = (47.241 + 51.178)/2 nm = 49.21 nm Conclusion: The obtained diameter is too big for a single protein nanofiber which is about 10-15 nm. It seems that 4-6 single fibers have bundled together.

  9. Results: Mean Diameter of Poly Q fibre 2 = (51.178 + 51.178)/2 nm = 51.178 nm Conclusion: The obtained diameter is too big for a single protein nanofiber which is about 10-15 nm. It seems that 4-6 single fibers have bundled together.

  10. Determination of length Results: Length of Poly Q fibre 1 = 175.78 nm Length of Poly Q fibre 2 = 164.06 nm Length of Prionfibre = 386.72 nm Conclusion: Poly Q fibre is shorter than Prionfibre which is perhaps because Glutamine (constituent of Poly Q) is a very simple molecule with less binding affinity. Prions are more prone to growth and binding to its own type.

  11. Force-distance curve in Peak-force tapping mode • a) The graph of the AFM tip trajectory over time during a single approach–retract cycle. (A): the tip is far off the surface and is lowered to the sample, going through an attractive field first until it reaches the contact point (B), after which the tip experiences a repulsive field as it begins to indent into the sample until it reaches the maximum Peak Force (C) that is used for the feedback (or to trigger the retraction portion of the cycle). In the second part of the cycle, the tip starts pulling off until it reaches its maximum adhesion (D) and finally recovers back its original position (E and A). • b) The maximum peak force curve (force versus distance) is used to extract the different properties of material. The adhesion force is calculated as in regular force mode and is the maximum step height between the baseline and the minimum value of the curve. The deformation is calculated as the distance between the contact point on the adhesion curve and the maximum indentation projected on the x axis. The dissipation is obtained by integrating the area between the extension and retraction curves. The elastic modulus is calculated by extrapolating the retraction curve close to the contact point and using a DMT/ JKR fit.

  12. REFERENCES • Lijiang; “Investigating Self-Assembled Protein Nanotubes Using Atomic Force Microscopy.” (Ph.D Thesis submitted to the University of Nottingham in July 2009) • B.Cappella, G.Dietler; “Force-distance curves by atomic force microscopy”.Elsevier Surface Science Reports 34 (1999) 1-104. • H.J.Butt, B.Cappella, M.Kappl; “Force measurements with the atomic force microscope: Technique, interpretation and applications”. ElsevierSurface Science Reports 59 (2005). • AdamcikJozef, MezzengaRaffaele; “Study of Amyloid Fibrils via Atomic Force Microscopy”. Current Opinion in Colloid & Interface Science (8th August 2012), doi:10.1016/j.cocis.2012.08.001 • John van Noort; “Atomic force microscopy of dynamic protein DNA interactions.” University of Twente, Netherlands. (25th June 2012). • JulyA. Ravi Patel; "A Morphological Study of Nanomaterials and Biomolecules using Atomic Force Microscopy." (3rd August 2012). Open Access Theses. Paper 371. • Kim Sweers, Martin Bennink and Vinod Subramaniam; “Nanomechanical properties of single amyloid fibrils.” IOP Journal of physics: Condensed Matter 24 (2012). • Kim Sweers, Kees van der Werf, Martin Bennink and Vinod Subramaniam; “Nanomechanical properties of α-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic force microscopy and Peakforce QNM.” Nanoscale Research Letters 2011, 6:270. • T J Young, M A Monclus, T L Burnett, W R Broughton, S L Ogin and P A Smith; “The use of the PeakForceTM quantitative nanomechanical mapping AFM-based method for high-resolution Young’s modulus measurement of polymers.” IOP science 2011.

  13. REFERENCES(arranged in descending order of importance) • MultiMode™ SPM Instruction Manual Version 4.31ce (402 pages) • U. Hartmann; “An Elementary Introduction to Atomic Force Microscopy and Related Methods”. Institute of Experimental Physics, University of Saarbrücken. (1997). • Tomasz Kowalewski, David M Holtzman; “In situ atomic force microscopy study of Alzheimer’s β-amyloid peptide on different substrates: New insights into mechanism of β-sheet formation”.Proc. Natl. Acad. Sci. USAVol. 96, pp. 3688–3693, March 1999 Biophysics • Véronique Vié, Marie-Cécile G, Eric L, Eric Finot, Jean-Pierre G, Christian Le Grimellec; “Tapping-mode atomic force microscopy on intact cells: optimal adjustment of tapping conditions by using the deflection signal”. ElsevierUltramicroscopy 82 (2000) 279-288. • Ricardo García, Ruben Péréz; “Dynamic Force Microscopy Methods”. ElsevierSurface Science Reports 47 (2002) 197-301. • Greg Haugstad; “Atomic Force Microscopy: Understanding Basic Modes and Advanced Applications”. • Veeco Dimension 3100 Atomic Force Microscope Users Manual • G J Vansco, H Schönherr; “Atomic Force Microscopy in Practice” • AlexandreBerquand, Bruker Nano Surfaces; “Quantitative Imaging of Living Biological Samples by PeakForce QNM Atomic Force Microscopy”. (2011) • http://en.wikipedia.org/wiki/Contact_mechanics Analysis of the AFM images was done using Nanoscope 5.31r1 software. You can download all the references at this link: http://kbose.weebly.com/msc-project.html

  14. The Vecco Multimode AFM used (costs Rs. 2 crore)

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