Biochemistry of malaria parasite infected red blood cells by X-ray microscopy.

Red blood cells infected by the malaria parasite Plasmodium falciparum are correlatively imaged by tomography using soft X-rays as well as by scanning hard nano-X-ray beam to obtain fluorescence maps of various elements such as S and Fe. In this way one can deduce the amount of Fe bound either in hemoglobin or in hemozoin crystals in the digestive vacuole of the malaria parasite as well as determine the hemoglobin concentrations in the cytosols of the red blood cell and of the parasite. Fluorescence map of K shows that in the parasite's schizont stage the K concentration in the red blood cell cytosol is diminished by a factor of seven relative to a pristine red blood cell but the total amount of K in the infected red blood cell is the same as in the pristine red blood cell.

Observation of structural anisotropy and the onset of liquidlike motion during the nonthermal melting of InSb.

The melting dynamics of laser excited InSb have been studied with femtosecond x-ray diffraction. These measurements observe the delayed onset of diffusive atomic motion, signaling the appearance of liquidlike dynamics. They also demonstrate that the root-mean-squared displacement in the [111] direction increases faster than in the [110] direction after the first 500 fs. This structural anisotropy indicates that the initially generated fluid differs significantly from the equilibrium liquid.

Clocking femtosecond X rays.

Linear-accelerator-based sources will revolutionize ultrafast x-ray science due to their unprecedented brightness and short pulse duration. However, time-resolved studies at the resolution of the x-ray pulse duration are hampered by the inability to precisely synchronize an external laser to the accelerator. At the Sub-Picosecond Pulse Source at the Stanford Linear-Accelerator Center we solved this problem by measuring the arrival time of each high energy electron bunch with electro-optic sampling. This measurement indirectly determined the arrival time of each x-ray pulse relative to an external pump laser pulse with a time resolution of better than 60 fs rms.

Atomic-scale visualization of inertial dynamics.

The motion of atoms on interatomic potential energy surfaces is fundamental to the dynamics of liquids and solids. An accelerator-based source of femtosecond x-ray pulses allowed us to follow directly atomic displacements on an optically modified energy landscape, leading eventually to the transition from crystalline solid to disordered liquid. We show that, to first order in time, the dynamics are inertial, and we place constraints on the shape and curvature of the transition-state potential energy surface. Our measurements point toward analogies between this nonequilibrium phase transition and the short-time dynamics intrinsic to equilibrium liquids.

Cholesterol monohydrate nucleation in ultrathin films on water.

The growth of a cholesterol crystalline phase, three molecular layers thick at the air-water interface, was monitored by grazing incidence x-ray diffraction and x-ray reflectivity. Upon compression, a cholesterol film transforms from a monolayer of trigonal symmetry and low crystallinity to a trilayer, composed of a highly crystalline bilayer in a rectangular lattice and a disordered top cholesterol layer. This system undergoes a phase transition into a crystalline trilayer incorporating ordered water between the hydroxyl groups of the top and middle sterol layers in an arrangement akin to the triclinic 3-D crystal structure of cholesterol x H(2)O. By comparison, the cholesterol derivative stigmasterol transforms, upon compression, directly into a crystalline trilayer in the rectangular lattice. These results may contribute to an understanding of the onset of cholesterol crystallization in pathological lipid deposits.

Crystalline films of interdigitated structures formed via amidinium-carboxylate interactions at the air-water interface.

Electrostatic interactions between amidinium and carboxylates were used for the construction of interdigitated architectures at the air-solution interface. Spreading the water-insoluble amphiphile p-pentadecylbenzoic acid (A) on an aqueous solution of p-methylbenzamidinium (B) ions results in an intercalation of the water-soluble base between the acidic headgroups of the water-insoluble amphiphile to form an amorphousA-B-A-B monolayer according to grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity measurements. Upon compression the monolayer transforms into a crystalline film composed of three bilayers with interdigitated hydrocarbon chains, and a top layer whose chains are disordered. Water-insoluble p-heptadecylbenzamidinium spread on an aqueous solution of benzoic acid displays a surface pressure-area isotherm similar to that obtained from the above system. A mechanism that accounts for the formation of these films is presented. Deposition of p-heptadecylbenzamidinium and p-pentadecylbenzoic acid amphiphiles in a 1:1 ratio on pure water led to the formation of a crystalline monolayer phase but which is partially disordered. Over an aqueous solution containing a 1:1 mixture of benzamidinium and benzoic acid no measurable binding of these solute molecules to the polar headgroups of the 1:1 mixed monolayer could be detected by X-ray reflectivity or GIXD.

Crystalline self-assembly into monolayers of folded oligomers at the air-water interface

Insertion of the 1,3-bis(ethynylene)benzene unit as a rigid spacer into a linear alkyl chain, thus separating the two resulting stems by 9 A. induces chain folding at the air-water interface. These folded molecules self-assemble into crystalline monolayers at this interface, with the plane of the folding unit almost perpendicular to the water surface, as determined by synchrotron grazing-incidence X-ray diffraction. Three distinct molecular shapes, of the types U, inverted U, and M, were obtained in the two-dimensional crystalline state, depending upon the number of spacer units, and the number and position of the hydrophilic groups in the molecule. The molecules form ribbons with a higher crystal coherence in the direction of stacking between the molecular ribbons, and a lower coherence along the ribbon direction. A similar molecule, but with a spacer unit that imposes a 5 A separation between alkyl chains, yields the conventional herringbone arrangement.

Oriented crystalline monolayers and bilayers of 2 x 2 silver(I) grid architectures at the air-solution interface: their assembly and crystal structure elucidation

Oriented crystalline monolayers, approximately 14 A thick, of a 2 x 2 Ag+ grid complex, self-assembled at the air-solution interface starting from an water-insoluble ligand 3,6-bis[2-(6-phenylpyridine)]pyridazine spread on silver-ion-containing solutions, were examined by grazing-incidence X-ray diffraction and specular X-ray reflectivity using synchrotron radiation. The monolayer structure was refined, including a determination of the positions of the counter-ions, with the SHELX-97 computer program. The monolayers were transferred from the interface onto various solid supports and visualized by scanning force microscopy, and characterized by X-ray photoelectron spectroscopy in terms of molecular structure. On surface compression, the initial self-assembled monolayer undergoes a transition to a crystalline bilayer in which the two layers, almost retaining the original arrangement, are in registry. Such a phase transition is of relevance to the understanding of crystal nucleation.

Formation of Chiral Interdigitated Multilayers at the Air-Liquid Interface Through Acid-Base Interactions

Thin interdigitated films composed of a long-chain, water-insoluble chiral acid (p-pentadecylmandelic acid of absolute configuration R) and a water-soluble chiral base (phenylethylamine, R') were constructed at the air-solution interface. The (R, R') structure was characterized to near-atomic resolution by grazing-incidence x-ray diffraction (GIXD). The two diastereomeric systems, (R, R') and (R, S'), demonstrate similar surface pressure-molecular area isotherms, but their structures are completely different on the molecular level, as monitored by GIXD. Complementary data on these two architectures were provided by atomic force microscopy.

Structural basis of cell-cell adhesion by cadherins.

Crystal structures of the amino-terminal domain of N-cadherin provide a picture at the atomic level of a specific adhesive contact between cells. A repeated set of dimer interfaces is common to the structure in three lattices. These interactions combine to form a linear zipper of molecules that mirrors the linear structure of the intracellular filaments with which cadherins associate. This cell-adhesion zipper may provide a mechanism to marshal individual molecular adhesive interactions into strong bonds between cells.

Control of structure and growth of polymorphic crystalline thin films of amphiphilic molecules on liquid surfaces.

The spontaneous formation and coexistence of crystalline polymorphic trilayer domains in amphiphilic films at air-liquid interfaces is demonstrated by grazing incidence synchrotron x-ray diffraction. These polymorphic crystallites may serve as models for the early stages of crystal nucleation and growth, helping to elucidate the manner in which additives influence the progress of crystal nucleation, growth, and polymorphism and suggesting ways of selectively generating and controlling multilayers on liquid surfaces. Auxiliary molecules have been designed to selectively inhibit development of the polymorphs, leading primarily to a single phase monolayer.

Langmuir-blodgett films of a functionalized molecule with cross-sectional mismatch between head and tail.

A functionalized surfactant has been investigated as floating monolayers by synchrotron x-ray diffraction and as bilayers transferred to solid supports by the Langmuir-Blodgett technique through atomic force microscopy. The transfer process is accompanied by an increase of the unit cell area (about 17 percent) and by an increase of the average domain diameter of nanometer-scale domains (about three times). The unit cell area of the floating monolayer corresponds to close packing of the head groups and a noncharacteristic packing of the tifted alkyl chains. The larger unit cell area of the bilayer film is consistent with a particular ordered packing of the alkyl chains, leaving free space for the head groups.

Crystal structures at 2.5 angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate.

Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 A resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine side chain, the interactions are likely to be similar in all class 2 synthetases.

Phases of phosphatidyl ethanolamine monolayers studied by synchrotron x-ray scattering.

For the first time, phospholid monolayers at the air/water interface have been studied by x-ray diffraction and reflection all along the isotherm from the laterally isotropic fluid (the so-called LE phase) to the ordered phases. The model used to analyze the data, and the accuracy of the parameters deduced, were tested by comparing the results obtained with two lipids having the same head group but different chain lengths. Compression of the fluid phase leads predominantly to a change of thickness of the hydrophobic moiety, much less of its density, with the head group extension remaining constant. The main transition involves a considerable increase (approximately 10%) of the electron density in the hydrophobic region, a dehydration of the head group and a positional ordering of the aliphatic tails, albeit with low coherence lengths (approximately 10 spacings). On further compression of the film, the ordered phase undergoes a continuous transition. This is characterized by an increase in positional ordering, a discontinuous decrease in lateral compressibility, a decrease in chain tilt angle with respect to the surface normal towards zero and probably also a head group dehydration and ordering.

Recognition processes at a functionalized lipid surface observed with molecular resolution.

The specific binding of proteins to functionalized lipid monolayers on aqueous subphases was characterized by neutron reflectivity and fluorescence microscopy measurements. Due to the high affinity and high specificity of their noncovalent interaction, streptavidin (SA) and biotin (vitamin H) were chosen as a model system to investigate the structural characteristics of a recognition process on a molecular length scale. Changes in the neutron reflection from the surfaces of NaCl aqueous (H2O or D2O) protein solutions (10(-8) M SA) were used to monitor the interaction of the protein with a monolayer of a biotinylated lipid in situ. Refinement of the reflectivity data and independent fluorescence microscopic observation of the interface using FITC-labeled SA showed that the protein forms macroscopically homogeneous (and presumably crystalline) domains covering a large portion of the surface. Moreover, the neutron reflection experiments clearly showed the formation of a monomolecular protein layer with an effective thickness, dp = 43.7 +/- 2 A. The area per protein molecule occupied in the film was A0 = 2860 +/- 200 A2 and nw = 260 +/- 100 water molecules were associated with each protein molecule. Quantitative binding was found to occur at biotin surface concentrations as low as 1 molecule/1,250 A2 (compared with approximately 1 molecule/40 A2 for dense packing). This study demonstrates the application of a promising new tool for the systematic investigation of molecular recognition processes in protein/lipid model systems.