High-pressure small-angle X-ray scattering cell for biological solutions and soft materials

Abstract

Pressure is a fundamental thermodynamic parameter controlling the behavior of biological macromolecules. Pressure affects protein denaturation, kinetic parameters of enzymes, ligand binding, membrane permeability, ion transduction, expression of genetic information, viral infectivity, protein association and aggregation, and chemical processes. In many cases pressure alters the molecular shape. Small-angle X-ray scattering (SAXS) is a primary method to determine the shape and size of macromolecules. However, relatively few SAXS cells described in the literature are suitable for use at high pressures and with biological materials. Described here is a novel high-pressure SAXS sample cell that is suitable for general facility use by prioritization of ease of sample loading, temperature control, mechanical stability and X-ray background minimization. Cell operation at 14 keV is described, providing a q range of 0.01 < q < 0.7 Å−1, pressures of 0–400 MPa and an achievable temperature range of 0–80°C. The high-pressure SAXS cell has recently been commissioned on the ID7A beamline at the Cornell High Energy Synchrotron Source and is available to users on a peer-reviewed proposal basis.

In Situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress

“…This can potentially provide insights into the influence of protein thermodynamic stability under cold-temperature conditions. Rosa et al proposed that the pressure effect on a model globular protein (bovine hemoglobin) in the cold-temperature regime was negligible by performing isochoric experiments ( ex situ approach) . Both globular proteins tested here have shown to be resistant to pressures up to 3 kbar under low-temperature conditions, which is higher than the pressure range associated with isochoric methods (pressure increases up to ∼1.5 kbar at −15 °C).…”

“…Rosa et al proposed that the pressure effect on a model globular protein (bovine hemoglobin) in the cold-temperature regime was negligible by performing isochoric experiments (ex situ approach). 80 Both globular proteins tested here have shown to be resistant to pressures up to 3 kbar under low-temperature conditions, which is higher than the pressure range associated with isochoric methods (pressure increases up to ∼1.5 kbar at −15 °C). Instead, Ova structural changes and the first protein− protein assembly steps detected via in situ HP-SANS below −10 °C are assumed to be induced solely by cold-temperature conditions.…”

“…Preliminary studies were also carried out using a custom-made HP cell for SAXS, at the Cornell High Energy Synchrotron Source (CHESS). The SAXS HP cell design and corresponding protein studies under pressure environment have been described elsewhere. − The results obtained from SAXS runs show that no significant conformational changes can be detected for Ova and aCgn (prepared in the corresponding H 2 O-based buffer) at a temperature range of ∼20 to −7 °C and pressures up to ∼3 kbar (data not shown). A lower-temperature range was not possible to test with the available SAXS HP cell.…”

Inline SAXS-coupled chromatography under extreme hydrostatic pressure

“…Glucose isomerase, a well-studied tetrameric protein standard for SAXS, is structurally stable at pressures up to at least 300 MPa 11 . Figure 1A compares elution profiles of a 100 µl injection of concentrated glucose isomerase (17.9 mg/ml) at room pressure and at 102.3 MPa ( P inlet ).…”

“…Glucose isomerase used in this study was from the same batch as used in evaluating our static HP-SAXS system. The preparation protocol can be found in the paper describing that work 11 .…”