High Pressure Solution SAXS

Nozomi Ando, Sol GrunerCornell University, Ithaca, NY 14853

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: 1http://www.volvox.co.jp/b_pcat/b04003.html

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. Jellyfish2. It is believed that life on earth, starting from the fundamental molecules of life, originated in the sea.

Photo: 2http://www.ucmp.berkeley.edu/cnidaria/scyphozoa.html

Features of High Pressure SAXS Cell

A. High pressure cell

• use of commercially available and custom-made high pressure parts2 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 holes3

• once set up, windows are in place and are never moved, thus keeping the sample thickness the same between sample and background measurements

3K. 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

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)

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)

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).