1. High Pressure Solution SAXS Nozomi Ando, Sol Gruner Cornell University, Ithaca, NY 14853

2. Motivation for High Pressure Solution SAXS Why pressure? There are many reasons to study systems using pressure as the perturbant. Many microorganisms exist in extreme conditions. Life can exist in pressures up to 1000 times atmospheric pressure. There are many mysteries yet to be understood in the realm of high pressure. Strange behavior can occur when pressure is increased, such as protein unfolding and polymer gel swelling. Extensive work has been done with temperature as a thermodynamic variable. Further studies of pressure will bring new insights and a more complete understanding of systems. In some ways, pressure is easier to manipulate than temperature. For example, a pressure jump can be easily achieved with pressure. Unlike chemical perturbants, pressure does not introduce anything new into the system. This is an attractive means for studying protein unfolding processes, which would otherwise require chemical perturbation. Photos on title page and this page: 1 http://www.volvox.co.jp/b_pcat/b04003.html

3. Small Angle X-ray Scattering as a Probe Why are we interested in studying solution samples with SAXS technique? Whether we study proteins or synthetic polymers, ultimately, we are interested in understanding biological systems. Structure, conformation of biomaterials function, behavior. SAXS is a powerful method for probing structural information of materials in solution. Figure 1. Jellyfish 2. It is believed that life on earth, starting from the fundamental molecules of life, originated in the sea. Photo: 2 http://www.ucmp.berkeley.edu/cnidaria/scyphozoa.html

4. Features of High Pressure SAXS Cell A. High pressure cell use of commercially available and custom- made high pressure parts 2 manufactured by High Pressure Equipment Co. (PA, USA) B. Windows use of diamond for low Z and high tensile strength, replacing traditional use of Be, which is toxic as an oxide natural diamonds originally used for diamond anvil cells were polished down to disks (William Bassett, Cornell University) use of Bridgeman type seal to seal windows onto modified plugs with holes 3 once set up, windows are in place and are never moved, thus keeping the sample thickness the same between sample and background measurements 3 K. Pressl et al, Rev. Sci. Instrum., 68, 12, 4588-4592 Figure 2. Diamond window. (a) Use of gem-shaped diamonds. (b) Bridgeman type seal utilized for sealing diamonds onto modified plugs. (a) (b) Bridgeman seals

5. High Pressure Solution SAXS Cell Figure 3. SAXS cell mounted onto thermostatic cell holder. Sample exchanged from top, high pressure fed from bottom of cell. (a) collimated X-ray enters cell though front diamond window and (b) exits back diamond window with scattering angle. (a)(b)

6. Applications Some materials of interest… Biological Materials –proteins –DNA –lipids –cells, viruses Synthetic Materials –polymers –polymer gels Figure 4. Applications (a) www.lbl.gov/enews/2-15-02.html (b) web.mit.edu/physics/tanaka/ (c) www.tulane.edu/~dmsander/Big_Virology/BVHomePage.html (a)(c) (b)

7. Pressure Effects on Transthyretin Transthyretin is one protein of interest. Transthyretin (TTR): –thyroxine, retinol-binding protein –55,000 Da tetramer –aggregation causes amyloidogenic diseases Aggregation of TTR can be induced by pH shift, hydrostatic pressure, temperature. Ferrao-Gonzales et al. studied preaggregated “intermediate” state. Free energy landscape can be studied using high pressure solution SAXS. aggregation T or P denatured Figure 5. Ferrao-Gonzales et al. PNAS, 97, 6445-6450 (2000).