Right Angle Light Scattering

Right angle light scattering (RALS) is an easy technique to perform using a standard spectrofluorimeter. It is used in this way mainly in the life sciences to follow the kinetics of reactions. Marrington et al. have used this technique

(a)

coupled with LD to follow the assembly of the bacterial protein FtsZ into a polymer and the subsequent bundling.25

They were able to measure the polymerization kinetics and the effects of Ca2+

and Mg2+

on this process by using RALS. It was also used by Mayer and Amann to monitor the polymerization of the bacterial actin MreB, and the effect of concentration on this process.26

It is an attractive technique because it is very simple to carry out, and can be used on a variety of sizes and shapes of particles.

In this work a Jasco FP-600 spectrofluorometer was used for RALS measurements. It has a Peltier cell attachment, which was used for temperature control during experiments. The cuvette used for RALS in this work was 1 cm ×

1 cm which is much bigger than the LD capillary. This means that there are no issues with the fibres becoming as large as the vessel holding them, and there is no problem with orientation as there is in LD, because RALS does not require particles to be oriented. Monochromatic light is shone onto the sample and the intensity of this light scattered at 90 ° is detected. This is very quick because only one wavelength is used per time point, rather than the whole spectrum as in LD. Therefore, the temporal resolution is much better than LD and a measurement was taken every second. As the size of the particles increases, the amount of light scattered also increases, so we can monitor the growing fibres. This is useful as a complementary technique to LD as, it allows us to probe the small fibres at the early stages which are invisible to LD, as well as probing large fibres that are so big that they get stuck in the narrow LD capillary.

An angle of 90 ° is ideal for light scattering measurements as this avoids the detection of light which passes straight through the sample, without interacting. Also by using a wavelength of light that is not absorbed by the sample, we avoid detecting emitted light, and instead, only detect light which has scattered. In this case 350 nm was used as FF does not absorb light at this wavelength.

The drawback of RALS is that although it is possible to detect that the size of the particles is increasing, the nature of the particle is unclear. It is necessary to use another technique, in this case LD or microscopy, to check that it is fibres that

are present, and not another large shape such as spheres or amorphous aggregates. It is possible to calculate the size of particles based on the light scattering intensity, if the shape of the particles is known and the sample is homogenous. However, the fibres were found to be heterogeneous so it was impossible to extract quantitative data about their size without using another technique, such as microscopy.

References

[1] Tkaczyk, T. C. (2010). Field Guide to Microscopy. Spie

[2] Shong, C. W., Haur, S. C., & Wee, A. T. S (2010). Science at the Nanoscale. Pan Stanford Publishing.

[3] Wöhler, C. (2013). 3D Computer Vision. Efficient Methods and Applications. Second Edition. Springer.

[4] Wittke, J. H. (2008). www4.nau.edu/microanalysis/microprobe- sem/signals.html. Accessed 16/03/2015

[5] Egerton, R. F. (2005). Physical Principles of Electron Microscopy. Springer

[6] Hunter, E. E. (1993). Practical Electron Microscopy: A Beginner’s Illustrated Guide. Cambridge University Press

[7] Willaims, D. and Carter, B. (2009). Transmission Electron Microscopy: A Textbook for Materials Science. Springer

[8] Grotendorst, J., Marx, D., & Muramatsu, A. (2002). Quantum Simulations of Complex Many-Body Systems: From Theory to Algorithms. NIC Directors.

[9] Griebel, M., Knapek, S., & Zumbusch, G. (2000). Numerical Simulation in Molecular Dynamics. Springer.

[10] Schachinger, E., & Stickler, B. A. (2014). Basic Concepts in Computational Physics. Springer.

[11] Phillips, J.C. et al. (2005). Scalable Molecular Dynamics with NAMD. J.

Comput. Chem.,26(16), 1781-1802.

[12] Frenkel, D., & Smit, B. (2002). Understanding Molecular Simulation.

Academic Press.

[13] Brooks, B. R., Brooks III, C. L., Mackerell, A. D., Nilsson, L., Petrella, R., J., Roux, B., Won, Y., …Karplus, M. (2009). CHARMM: The biomolecular simulation program. J. Comp. Chem., 30, 1545-1615.

[14] Vymetal, J. & Vondrášek, J. (2010). Metadynamics As a Tool for Mapping the Conformational and Free-Energy Space of Peptides – The Alanine Dipeptide Case Study. J. Phys. Chem. B, 114(16), 5632-5642.

[15] König, G., Bruckner, S. and Boresch, S. (2013). Absolute Hydration Free Energies of Blocked Amino Acids: Implications for Protein Solvation and Stability. Biophys. J., 104, 453-462.

[16] Allen, F. H. (2002). The Cambridge Structural Database: a Quarter of a Million Crystal Structures and Rising. Acta Cryst., B58, 380-388.

[17] Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., & Ferrin, T. E. (2004). UCSF Chimera—A Visualisation System for Exploratory Research and Analysis. J. Comput. Chem., 26(16), 1781- 1802.

[18] Ferrario, M., Ciccotti, G., & Binder, K. (2006). Computer Simulations in Condensed Matter Systems: From Materials to Chemical Biology. Springer.

[19] Bonomi, M et al. (2009). PLUMED: a portable plugin for free-energy calculations with molecular dynamics. Comp. Phys. Chem., 180, 1961-1975. [20] Platt, Y., & Stutz, J. (2008). Physics of Earth and Space Environments.

Springer.

[21] Norden, B., Rodger, A., & Dafforn, T. (2010). Linear Dichroism and Circular Dichroism. RSC Publishing.

[22] Dafforn, T., Rajendra, J., Halsall, D. J., Serpell, L. C., & Rodger, A. (2004). Protein Fiber Linear Dichroism for Structure Determination and Kinetics in a Low-Volume, Low-Wavelength Couette Flow Cell. Biophysical Journal, 8, 404- 410.

[23] Cheng, X., Joseph, M. B., Covington, J. A., Dafforn, T. R., Hicks, M. R., & Rodger, A. (2012). Continuous-Channel Flow Linear Dichroism. Analytical

Methods, 4, 3169-3173.

[24] Marshall, K. E., Hicks, M. R., Williams, T. L., Hoffmann, S. V., Rodger, A., Dafforn, T. R., & Serpell, L. C. (2010). Characterizing the Assembly of the Sup35 Yeast Prion Fragment, GNNQQNY: Structural Changes Accompany a Fiber-to-Crystal Switch. Biophysical Journal, 98, 330-338.

[25] Marrington, R., Small, E., Rodger, A., Dafforn, T. R., & Adinall, S. G. (2004). FtsZ Fiber Bundling Is Triggered by a Conformational Change in Bound GTP. The Journal of Biological Chemistry. 279 (47), 48821-48829.

[26] Mayer, J. A., & Amann, K. J. (2009) Assembly Properties of the Bacillus subtilis Actin, MreB. Cell Motiliy and the Cytoskeleton, 66, 109-118.

Chapter 3