High-pressure Small-angle X-ray Research Articles

The effect of hydrostatic pressure on lipid membrane lateral structure.

High pressure is both an environmental challenge to which deep sea biology has to adapt, and a highly sensitive thermodynamic tool that can be used to trigger structural changes in biological molecules and assemblies. Lipid membranes are amongst the most pressure sensitive biological assemblies and pressure can have a large influence on their structure and properties. In this chapter, we will explore the use of high pressure small angle X-ray diffraction and high pressure microscopy to measure and quantify changes in the lateral structure of lipid membranes under both equilibrium high pressure conditions and in response to pressure jumps.

Protein unfolded states populated at high and ambient pressure are similarly compact

The relationship between the dimensions of pressure-unfolded states of proteins compared with those at ambient pressure is controversial; resolving this issue is related directly to the mechanisms of pressure denaturation. Moreover, a significant pressure dependence of the compactness of unfolded states would complicate the interpretation of folding parameters from pressure perturbation and make comparison to those obtained using alternative perturbation approaches difficult. Here, we determined the compactness of the pressure-unfolded state of a small, cooperatively folding model protein, CTL9-I98A, as a function of temperature. This protein undergoes both thermal unfolding and cold denaturation, and the temperature dependence of the compactness at atmospheric pressure is known. High-pressure small angle x-ray scattering studies, yielding the radius of gyration and high-pressure diffusion ordered spectroscopy NMR experiments, yielding the hydrodynamic radius were carried out as a function of temperature at 250 MPa, a pressure at which the protein is unfolded. The radius of gyration values obtained at any given temperature at 250 MPa were similar to those reported previously at ambient pressure, and the trends with temperature are similar as well, although the pressure-unfolded state appears to undergo more pronounced expansion at high temperature than the unfolded state at atmospheric pressure. At 250 MPa, the compaction of the unfolded chain was maximal between 25 and 30°C, and the chain expanded upon both cooling and heating. These results reveal that the pressure-unfolded state of this protein is very similar to that observed at ambient pressure, demonstrating that pressure perturbation represents a powerful approach for observing the unfolded states of proteins under otherwise near-native conditions.

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

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 trans-duction, 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.

Structural changes in lipid mesophases due to intercalation of dendritic polymer nanoparticles: Swollen lamellae, suppressed curvature, and augmented structural disorder

Understanding interactions between nanoparticles and model membranes is relevant to functional nano-composites and the fundamentals of nanotoxicity. In this study, the effect of polyamidoamine (PAMAM) dendrimers as model nanoparticles (NP) on the mesophase behaviour of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) has been investigated using high-pressure small-angle X-ray scattering (HP-SAXS). The pressure-temperature (p−T) diagrams for POPE mesophases in excess water were obtained in the absence and presence of G2 and G4 polyamidoamine (PAMAM) dendrimers (29 Å and 45 Å in diameter, respectively) at varying NP-lipid number ratio (ν = 0.0002–0.02) over the pressure range p = 1–3000 bar and temperature range T = 20–80 °C. The p−T phase diagram of POPE exhibited the Lβ, Lα and HII phases. Complete analysis of the phase diagrams, including the relative area pervaded by different phases, phase transition temperatures (Tt) and pressures (pt), the lattice parameters (d-spacing), the pressure-dependence of d-spacing (Δd/Δp), and the structural ordering in the mesophase as gauged by the Scherrer coherence length (L) permitted insights into the size- and concentration-dependent interactions between the dendrimers and the model membrane system. The addition of dendrimers changed the phase transition pressure and temperature and resulted in the emergence of highly swollen lamellar phases, dubbed Lβ-den and Lα-den. G4 PAMAM dendrimers at the highest concentration ν = 0.02 suppressed the formation of the HII phase within the temperature range studied, whereas the addition of G2 PAMAM dendrimers at the same concentration promoted an extended mixed lamellar region in which Lα and Lβ phases coexisted. Statement of SignificanceUsing high pressure small angle X-ray scattering in the pressure range 1–3000 bar and temperature range 20–60 °C, we have studied interactions between PAMAM dendrimers (as model nanoparticles) and POPE lipid mesophases (as model membranes). We report the pressure-temperature phase diagrams for the dendrimer-lipid mesophases for the first time. We find that the dendrimers alter the phase transition temperatures (Tt) and pressures (pt), the lattice parameters (d-spacing), and the structural order in the mesophase. We interpret these unprecedented results in terms of the fluidity of the lipid membranes and the interactions between the dendrimers and the membranes. Our findings are of fundamental relevance to the field of nanotoxicity and functional nanomaterials that integrate nanoparticles and organized lipid structures.

Pressure-Effects and Adaptation Mechanisms of Ambient and Deep-Sea Bacterial Enzymes

Submersible expeditions into the deepest parts of Earth's oceans have led to discoveries of organisms thriving in the extremes, raising questions on the limits of life and the functioning of their central biological macromolecules. Questions arise as to how life would be able to survive under such “harsh” conditions, and what adaptations may be present in these organisms’ proteins. Our major focus is on understanding biophysical effects of extreme pressure, which is a relatively underexplored condition for protein solutions. Investigating the effects of high hydrostatic pressure (HHP) on the biophysical properties of proteins, in conjunction with structural adaptations utilized by known piezophilic organisms to maintain proper function, provide better insight into how organisms are affected by and survive at such depths. The results gathered from these data can impact our use of pressure for sterilization of food products, our understanding of piezophilic adaptations, and enhance our understanding of the origins of life. Graphics processing unit (GPU)-accelerated OpenMM molecular dynamics simulations were used to explore pressure-effects on and pressure-adaptations within a central metabolic enzyme, dihydrofolate reductase (DHFR). In-silico comparisons of hydrogen bonding, collective motion, among other phenomena, were made between bacterial DHFRs of the mesophile (ambient pressure) Escherichia coli, the deep-sea piezophiles Moritella profunda and Moritella yayanosii, as well as mutants containing specific residues identified to be important in pressure-sensitivity. Furthermore, high-pressure small-angle neutron and X-ray scattering, fluorescence and other high-pressure experimental techniques conducted up to 3.5 kbar further probed the biophysical effects of HHP on E. coli and M. profunda DHFRs. The combined results from in-silico and in-situ techniques provide enhanced insight into the interplay between the sequence, structure, and hydration of proteins underpinning life inhabiting extreme pressures.

The consequences of cavity creation on the folding landscape of a repeat protein depend upon context

The effect of introducing internal cavities on protein native structure and global stability has been well documented, but the consequences of these packing defects on folding free-energy landscapes have received less attention. We investigated the effects of cavity creation on the folding landscape of the leucine-rich repeat protein pp32 by high-pressure (HP) and urea-dependent NMR and high-pressure small-angle X-ray scattering (HPSAXS). Despite a modest global energetic perturbation, cavity creation in the N-terminal capping motif (N-cap) resulted in very strong deviation from two-state unfolding behavior. In contrast, introduction of a cavity in the most stable, C-terminal half of pp32 led to highly concerted unfolding, presumably because the decrease in stability by the mutations attenuated the N- to C-terminal stability gradient present in WT pp32. Interestingly, enlarging the central cavity of the protein led to the population under pressure of a distinct intermediate in which the N-cap and repeats 1-4 were nearly completely unfolded, while the fifth repeat and the C-terminal capping motif remained fully folded. Thus, despite modest effects on global stability, introducing internal cavities can have starkly distinct repercussions on the conformational landscape of a protein, depending on their structural and energetic context.

Long Periodic Structure of a Room-Temperature Ionic Liquid by High-Pressure Small-Angle X-Ray Scattering and Wide-Angle X-Ray Scattering: 1-Decyl-3-Methylimidazolium Chloride.

The Bragg reflections of 1-decyl-3-methylimidazolium chloride ([C10 mim][Cl]), a room-temperature ionic liquid, are observed in a lowly scattered wavevector (q) region using high-pressure (HP) small-angle X-ray scattering methods. The HP crystal of [C10 mim][Cl] was characterized by an extremely long periodic structure. The peak position at the lowest q (1.4 nm-1 ) was different from that of the prepeak observed in the liquid state (2.3 nm-1 ). Simultaneously, Bragg reflections at high-q were detected using HP wide-angle X-ray scattering. The longest lattice constant was estimated to be 4.3 nm using structural analysis. The crystal structure of HP differed from that of the low-temperature (LT) crystal and the LT liquid crystal. With increasing pressure, Bragg reflections in the high-q component became much broader, and were accompanied by phase transition, although those in the low-q component were observed to be relatively sharp.

Tuning the Interaction of Nanoparticles from Repulsive to Attractive by Pressure

We report a high pressure small-angle X-ray scattering (SAXS) study on concentrated aqueous suspensions of gold nanoparticles coated with a poly(ethylene glycol) (PEG)-based ligand shell. Analyzing the experimental SAXS data with a liquid-state theory approach, we find a pressure-induced transition of the interparticle interactions from repulsive to attractive in the pressure range from 1 to 4000 bar that also depends on the particle concentration. This transition is related to structural changes of the ligand shell which is compressed with increasing pressure. Our study highlights the important, yet unexplored role of pressure for the interaction of nanoparticle systems.

Colloidal crystallite suspensions studied by high pressure small angle x-ray scattering.

We report on high pressure small angle x-ray scattering on suspensions of colloidal crystallites in water. The crystallites made out of charge-stabilized poly-acrylate particles exhibit a complex pressure dependence which is based on the specific pressure properties of the suspending medium water. The dominant effect is a compression of the crystallites caused by the compression of the water. In addition, we find indications that also the electrostatic properties of the system, i.e. the particle charge and the dissociation of ions, might play a role for the pressure dependence of the samples. The data further suggest that crystallites in a metastable state induced by shear-induced melting can relax to a similar structural state upon the application of pressure and dilution with water. X-ray cross correlation analysis of the two-dimensional scattering patterns indicates a pressure-dependent increase of the orientational order of the crystallites correlated with growth of these in the suspension. This study underlines the potential of pressure as a very relevant parameter to understand colloidal crystallite systems in aqueous suspension.

Stress-induced nanoparticle crystallization.

We demonstrate for the first time a new mechanical annealing method that can significantly improve the structural quality of self-assembled nanoparticle arrays by eliminating defects at room temperature. Using in situ high-pressure small-angle X-ray scattering, we show that deformation of nanoparticle assembly in the presence of gigapascal level stress rebalances interparticle forces within nanoparticle arrays and transforms the nanoparticle film from an amorphous assembly with defects into a quasi-single crystalline superstructure. Our results show that the existence of the hydrostatic pressure field makes the transformation both thermodynamically and kinetically possible/favorable, thus providing new insight for nanoparticle self-assembly and integration with enhanced mechanical performance.

Conformational changes upon high pressure induced hydration of poly(N-isopropylacrylamide) microgels

We investigated thermosensitive poly(N-isopropylacrylamide) microgels by high-pressure small angle X-ray scattering and Fourier-transform infrared spectroscopy below and above the collapse temperature. The measurements reveal little pressure-induced deswelling below the volume phase transition temperature and clear re-swelling of the collapsed gels at temperatures above the VPTT.

High-Pressure Structural Stability and Elasticity of Supercrystals Self-Assembled from Nanocrystals

We report here combined quasi-hydrostatic high-pressure small-angle X-ray scattering (SAXS) and X-ray diffraction (XRD) studies on faceted 3D supercrystals (SCs) self-assembled from colloidal 7.0 nm spherical PbS nanocrystals (NCs). Diamond anvil cell (DAC) SAXS experiments in the pressure range from ambient to 12.5 GPa revealed nearly perfect structural stability of the SCs, with face-centered cubic organization of the NCs. Pressure-induced ordering (annealing effect) of the superstructure was observed. The ambient pressure bulk modulus of the SCs was calculated to be ∼5 GPa for compression and ∼14.5 GPa for decompression from fitting of Vinet and Birch-Murnaghan equations of state. XRD measurements revealed strong preferential crystallographic orientation of the NCs through all phase transformations to as high as 55 GPa without any indication of NC sintering. The first phase transition pressure of the NCs was found between 8.1 and 9.2 GPa and proceeds through homogeneous nucleation. Bulk modulus of PbS NCs was calculated to be ∼51 GPa based on fitting to the equations of state (K(PbS,bulk) ∼ 51-57 GPa). Closest surface-to-surface distance between the NCs in the SCs was calculated based on combined XRD and SAXS data, to reversibly tune from ∼1.56 nm to ∼0.9-0.92 nm and back to ∼1.36 nm in the ambient-12.5 GPa-ambient pressure cycle. The bulk modulus of the ligand matrix was extrapolated to be ∼2.2-2.95 GPa. These results show a general method of tuning NC interactions in packed nanoparticle solids.

High-Pressure SAXS Study of Folded and Unfolded Ensembles of Proteins

A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.

Filament Structure, Organization, and Dynamics in MreB Sheets

In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several mum long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.

Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA

AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 Å, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 Å. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5′-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.

1P050 アクチン相同骨格タンパク質フィラメントMreB、AlfAの高圧X線小角散乱による研究(蛋白質-物性(安定性,折れたたみなど),第48回日本生物物理学会年会)

1P050 アクチン相同骨格タンパク質フィラメントMreB、AlfAの高圧X線小角散乱による研究(蛋白質-物性(安定性,折れたたみなど),第48回日本生物物理学会年会)

Transparency and structure of eye lens studied by high-pressure small-angle X-ray scattering

Transparency and structure of eye lens studied by high-pressure small-angle X-ray scattering

High hydrostatic pressure small-angle X-ray scattering cell for protein solution studies featuring diamond windows and disposable sample cells

A high-pressure cell for synchrotron small-angle X-ray scattering (SAXS) studies of protein solutions is described. The design was optimized for use at up to 400 MPa in liquid pressure and with 8−12 keV X-rays with particular emphasis on the ease of use. The high-pressure cell was fabricated from corrosion-resistant Inconel 725 (Special Metals Corporation, Huntington, WV, USA) and featured Poulter-type windows [Poulter (1932).Phys. Rev.40, 861–871]. Flat natural diamonds, 500 µm thick, were recycled from diamond anvil cells and were shown to perform well as high-pressure SAXS windows. For a simple and effective method of sample isolation, disposable plastic sample cells with a defined path length and reproducible parasitic scattering were designed. These sample cells enable efficient use of synchrotron time. The cells facilitate rapid and easy sample changes, eliminate the need to clean the cell between sample changes, and reduce the sample volume to as low as 12 µl. The disposable cells can also be used separately from the high-pressure cell for SAXS measurements at ambient pressure and temporary storage of samples. The performance of the apparatus is demonstrated with T4 lysozyme.

2P-342 高圧ジャンプX線小角散乱法を用いた細胞骨格フィラメントの安定性とヌクレオチドキャップの研究(バイオイメージング(2),第46回日本生物物理学会年会)

2P-342 高圧ジャンプX線小角散乱法を用いた細胞骨格フィラメントの安定性とヌクレオチドキャップの研究(バイオイメージング(2),第46回日本生物物理学会年会)

Met-myoglobin Association in Dilute Solution during Pressure-Induced Denaturation: an Analysis at pH 4.5 by High-Pressure Small-Angle X-ray Scattering

In this paper, we report on the original global fit procedure of synchrotron small-angle X-ray scattering (SAXS) data applied to a model protein, met-myoglobin, in dilute solution during temperature- and pressure-induced denaturation processes at pH 4.5. Starting from the thermodynamic description of the protein unfolding pathway developed by Hawley (Hawley, S. A. Biochemistry 1971, 10, 2436), we have developed a new method for analyzing the set of SAXS curves using a global fitting procedure, which allows us to derive the form factor of all the met-myoglobin species present in the solution, their aggregation state, and the set of thermodynamic parameters, with their p and T dependence. This method also overcomes a reasonably poor quality of the experimental data, and it is found to be very powerful in analyzing SAXS data. SAXS experiments were performed at four different temperatures from hydrostatic pressures up to about 2000 bar. As a result, the presence of an intermediate, partially unfolded, dimeric state of met-myoglobin that forms during denaturation has been evidenced. The obtained parameters were then used to derive the met-myoglobin p, T phase diagram that fully agrees with the corresponding phase diagram obtained by spectroscopic measurements.