Yeast-based production and purification of HIS-tagged human ATAD3A, A specific target of S100B.

ATAD3 is a mitochondrial integral inner membrane ATPase with unknown function. ATAD3 is absent in yeast and protozoan and present in all pluricellular eucaryotes where its expression is essential for development. To date, bacterial-based expression of full-length ATAD3 has been unsuccessful because of very high levels of endogenous degradation. Based on Saccharomyces cerevisiae as a heterogeneous expression system, we engineered a high copy strain expressing human ATAD3A-Myc-HIS at a relative high level (2.5mg/l of yeast culture) without significantly affecting yeast growth. Most of the expressed human ATAD3A-Myc-HIS co-purified with the yeast mitochondrial fraction thus suggesting that targeting to this organelle is preserved in yeast. Like the endogenous protein in human cells, ATAD3A-Myc-HIS expressed in yeast is found resistant to extraction with salt and certain detergents, suggesting membrane insertion. Sarkosyl, C13-DAO, C12-DAO and ONMG efficiently solubilized ATAD3A-Myc-HIS from yeast extracts, but these soluble species did not bind to agarose-nickel matrix. By contrast, urea-denaturated ATAD3A-Myc-HIS bound to agarose-nickel beads and could be renatured and eluted to obtain highly pure ATAD3A-Myc-HIS. As the native protein in vivo, this recombinant, renatured species specifically bound in vitro to S100B and S100A1 in Far-Western assays.

Conformational dynamics of the bovine mitochondrial ADP/ATP carrier isoform 1 revealed by hydrogen/deuterium exchange coupled to mass spectrometry.

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acid-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.

Effective removal of nonionic detergents in protein mass spectrometry, hydrogen/deuterium exchange, and proteomics.

Detergents are frequently used for protein isolation and solubilization. Their presence is crucial in membrane protein protocols or in lipid raft proteomics. However, they are usually poorly compatible with mass spectrometry. Several different sample preparation protocols are routinely used, but they are either laborious or suffer from sample losses. Here, we describe our alternative method for nonionic detergent removal. It is based on selective detergent extraction after capture of the sample on a reversed phase cartridge. The extraction is performed by chlorinated solvents and works well for polyoxyethylene based nonionic detergents, but also for polymers like polyethylene and propylene glycol. Detergent removal can be also carried out on the protein level but a special care must be taken with hydrophobic proteins. In such cases, it is preferable to perform detergent removal after proteolysis which digests the protein to peptides and reduces the hydrophobicity. The method can easily be automated and is compatible with hydrogen/deuterium exchange coupled to mass spectrometry.

Topological analysis of ATAD3A insertion in purified human mitochondria.

ATAD3 is a mitochondrial inner membrane-associated protein that has been predicted to be an ATPase but from which no associated function is known. The topology of ATAD3 in mitochondrial membranes is not clear and subject to controversy. A direct interaction of the N-terminal domain (amino-acids 44-247) with the mtDNA has been described, but the same domain has been reported to be sensitive to limited proteolysis in purified mitochondria. Furthermore, ATAD3 has been found in a large purified nucleoid complex but could not be cross-linked to the nucleoid. To resolve these discrepancies we used two immunological approaches to test whether the N-terminal (amino-acids 40-53) and the C-terminal (amino-acids 572-586) regions of ATAD3 are accessible from the cytosol. Using N-terminal and C-terminal specific anti-peptide antibodies, we carried out back-titration ELISA measurements and immuno-fluorescence analysis on freshly purified human mitochondria. Both approaches showed that the N-terminal region of ATAD3A is accessible to antibodies in purified mitochondria. The N-terminal region of ATAD3A is thus probably in the cytoplasm or in an accessible intermembrane space. On the contrary, the C-terminal region is not accessible to the antibody and is probably located within the matrix. These results demonstrate both that the N-terminal part of ATAD3A is outside the inner membrane and that the C-terminal part is inside the matrix.

Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.

ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved the bovine carrier structure at a resolution of 2.2 A by X-ray crystallography in complex with an inhibitor, carboxyatractyloside. Six alpha-helices form a compact transmembrane domain, which, at the surface towards the space between inner and outer mitochondrial membranes, reveals a deep depression. At its bottom, a hexapeptide carrying the signature of nucleotide carriers (RRRMMM) is located. Our structure, together with earlier biochemical results, suggests that transport substrates bind to the bottom of the cavity and that translocation results from a transient transition from a 'pit' to a 'channel' conformation.

Association of phosphatidic acid with the bovine mitochondrial ADP/ATP carrier.

The beef heart adenine nucleotide carrier protein (Anc) of the inner mitochondrial membrane can be purified in a form stabilized by binding the inhibitor carboxyatractyloside. The protein is copurified with bound lipid. We show for the first time that phosphatidic acid, although a minor component, is one of the lipids bound to Anc. The short spin-lattice relaxation time found by (31)P magic angle spinning nuclear magnetic resonance (MAS/NMR) for phosphatidic acid indicates that it is tightly bound to the protein. However, this lipid also has a comparatively small chemical shift anisotropy, suggesting that it can undergo rapid reorientation in space. In contrast, most of the lipid bound to Anc shows anisotropic motion typical of a bilayer arrangement. The phosphatidic acid that is detected in the purified preparation of Anc is also shown to be present initially in the unfractionated mitochondria, prior to the isolation of Anc. In Triton-solubilized mitochondria, phosphatidic acid, cardiolipin, phosphatidylethanolamine, and phosphatidylcholine exhibit resonance lines in the static (31)P NMR spectra, but in the purified Anc, only the phosphatidylethanolamine and phosphatidylcholine can be detected by this method, even though the other lipids are still present. This demonstrates that the phosphatidic acid and cardiolipin are interacting with the Anc. The thermal denaturation of the Anc was determined by differential scanning calorimetry. The protein denatures at 74 degrees C both before and after the NMR studies with the same characteristics.

Recombinant immobilized rhizopuspepsin as a new tool for protein digestion in hydrogen/deuterium exchange mass spectrometry.

Hydrogen/deuterium (H/D) exchange coupled to mass spectrometry is nowadays routinely used to probe protein interactions or conformational changes. The method has many advantages, e.g. very low sample consumption, but offers limited spatial resolution. One way to higher resolution leads through the use of different proteases or their combinations. In the present work we describe recombinant production, purification and use of aspartic protease zymogen from Rhizopus chimensis, protease type XVIII (EC 3.4.23.6), commonly referred to as rhizopuspepsinogen (Rpg). The enzyme was expressed in Escherichia coli, refolded and purified to homogeneity. A typical yield was approximately 100 mg of pure enzyme per 1 L of original bacterial culture. The kinetics of protease activation, i.e. removal of the propeptide achieved by autolysis in an acidic environment, was followed by mass spectrometry. The digestion efficiency was tested for the protease in solution as well as for the immobilized enzyme. Apomyoglobin was successfully digested under all conditions tested and the protease displayed very low or no autodigestion. The results outperformed those obtained with commercial protease where the digestion of apomyoglobin was incomplete and accompanied by many contaminating peptides. Taken together, the recombinant protease type XVIII can be considered as a new and highly efficient tool for H/D exchange followed by mass spectrometry.

Two residues of a conserved aromatic ladder of the mitochondrial ADP/ATP carrier are crucial to nucleotide transport.

The mitochondrial ADP/ATP carrier is the paradigm of the mitochondrial carrier family (MCF), whose members are crucial for cross-talks between mitochondria, where cell energy is mainly produced, and the cytosol, where cell energy is mainly consumed. These carriers share structural and functional characteristics. Resolution of the 3D structure of the beef mitochondrial ADP/ATP carrier, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in substrate binding and transfer from the outside to the inside of mitochondria. To ascertain the role of these residues, namely, Y186, Y190, F191, and Y194, they were mutated into alanine in the yeast mitochondrial ADP/ATP carrier at equivalent positions (Y203, Y207, F208, and Y211). Two residues, Y203 and F208, appeared to be crucial for transport activity but not for substrate binding per se, indicating their involvement in the substrate transfer process through the carrier. Furthermore, it was possible to show that these mutations precluded conformational changes of the matrix loop m2, whose movements were demonstrated to participate in substrate transport by the wild-type carrier. Therefore, these aromatic residues may be involved in substrate gliding, and they may also confer specificity toward adenine nucleotides for the ADP/ATP carrier as compared with the MCF members.

The Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein: one-step purification and functional analysis in vitro.

Genetic expression versus plasmidic overexpression of a functional recombinant fusion protein combining the yeast Saccharomyces cerevisiae mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p) has been investigated, with the main aim of increasing the polar surface of the carrier to improve its crystallization properties. The gene encoding the his6-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2 or under the control of the strong yeast PMA1 promoter. In both cases, the chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, demonstrating its transport activity. Nevertheless, when the expression vector was used, the level of expression of Anc2-Cyc1(His6)p was no greater than that of the chimeric carrier obtained in yeast mitochondria after homologous recombination. Optimal conditions to extract and to purify Anc2-Cyc1(His6)p were determined. A series of detergents was screened for their ability to extract and to preserve in vitro the chimeric carrier. A rapid, single step purification of Anc2-Cyc1(His6)p was developed, using n-dodecyl-beta-d-maltoside (DoDM) as the best detergent to solubilize the chimeric protein. Carboxyatractyloside- (CATR-) and nucleotide-binding sites were preserved in the purified protein. Moreover, the Cyc1p moiety of Anc2-Cyc1(His6)p-CATR complex solubilized in DoDM was still able to interact in vitro with the cytochrome c oxidase (COX), with the same affinity as yeast Cyc1p. Improved production and purification of Anc2-Cyc1(His6)p-CATR complex opens up new possibilities for the use of this protein in crystallographic approaches to the yeast ADP/ATP carrier. Furthermore, Anc2-Cyc1(His6)p may be an useful molecular tool to investigate in vivo interactions between components of the respiratory chain complexes such as COX and the proteins implicated in ATP biogenesis, such as the ATP/ADP carrier.

Mitochondrial carrier family: repertoire and peculiarities of the cellular slime mould Dictyostelium discoideum.

Proteins of the mitochondrial carrier family (MCF) mediate the transport of a large range of compounds, including metabolites and cofactors. They are localized mainly in the inner mitochondrial membrane, except for a few members found in the membranes of peroxisomes. Similarity searches among Dictyostelium discoideum protein sequences identified a total of 31 MCF members. All these are membrane proteins that possess three characteristic repeats of a domain of approximately 100 residues. Among them, three proteins have supplementary structural domains consisting of Ca(2+)-binding motifs made up of 2 or 4 EF-hand units localized on the N-terminal end, facing the mitochondrial intermembrane space. The nature of transported substrates is proposed on the basis of sequence comparison with orthologs characterized biochemically in other organisms, of phylogenetic analysis, and of the conservation of discriminating amino acid residues belonging to the substrate binding sites. Carriers have been grouped in subclasses based on their specificity for the transport of nucleotides, amino acids or keto acids. Furthermore, we have identified an iron carrier of the mitoferrin type, an inorganic phosphate carrier, and three carriers with similarity to uncoupler proteins. This study provides a focus for mitochondrial carrier analysis in Dictyostelium discoideum.

Nucleotide exchange in mitochondria: insight at a molecular level.

Mitochondrial carrier proteins are embedded in the inner mitochondrial membrane and ensure the transport of many important metabolites. The ADP/ATP carrier imports ADP into the mitochondrial matrix in exchange for ATP after synthesis. It is the most studied mitochondrial carrier and its structure was the first to be unraveled at high resolution. The structure reveals six transmembrane helices forming a tightly closed bundle toward the matrix and a funnel-shaped cavity opening toward the intermembrane space. The cavity ends in a narrow pit 10A from the matrix. The analysis of residues located in the cavity hints at the mechanism of binding of adenine nucleotides. Additionally, the presence of conserved proline residues in three sharply kinked helices suggests a translocation mechanism.

Native membrane proteins vs. yeast recombinant: an example: the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier (Ancp) has long been a paradigm for studies of the mitochondrial carrier family due to, among other properties, its natural abundance and the existence of specific inhibitors, namely, carboxyatractyloside (CATR) and bongkrekic acid (BA), which lock the carrier under distinct and stable conformations. Bovine Anc1p isolated in complex with CATR in the presence of an aminoxyde detergent (LAPAO) was crystallized and its 3D structure determined. It is the first mitochondrial carrier structure resolved at high resolution (2.2 A, as reported by Pebay-Peyroula et al. (Nature 426:39-44, 2003)). Analyses revealed a monomer while most of the biochemical studies led to hypothesize Ancp functions as a dimer. To address the structural organization issue, we engineered a mutant of the yeast Ancp that corresponds to a covalent homodimer in view of 3D structure determination. We compare in this chapter the purification yield and quality of the chimera tagged either with six histidines at its C-ter end or nine histidines at its N-ter. We show that, as expected, length and position of the tag are important criteria for qualitative purification. We also discuss the advantages and drawbacks of purifying Ancp either from a natural source or from engineered yeast cells.

The anti-inflammatory agent flufenamic acid depresses store-operated channels by altering mitochondrial calcium homeostasis.

Fenamates like flufenamic acid (FFA) are anti-inflammatory drugs known to alter ion fluxes through the plasma membrane. They are for instance potent blockers of cation and anion channels, and FFA is now commonly used to block currents through TRP channels and receptor-operated channels. However, FFA exerts complex and multifaceted actions on ion transport systems and, in most instances, a molecular understanding of these FFA-dependent modulations is lacking. In addition, FFA is also to known to perturb the homeostasis of Ca2+. In the present report, we investigated whether the FFA-induced alterations of the Ca2+ homeostasis could play a role in the FFA-dependent modulation of transmembrane ion fluxes. Experiments performed with the Ca2+ indicator Fluo-4 on cultured cortical neurons and HEK-293 cells showed that FFA increased the cytosolic concentration of Ca2+ even in cells kept in a Ca2+-free medium or when the endoplasmic reticulum was depleted with thapsigargin. The FFA-dependent Ca2+ responses were, however, strongly reduced by bongkrekic acid, a specific ligand of the mitochondrial ADP/ATP carrier which, in addition, inhibits the permeability transition pore. Like FCCP, FFA released Ca2+ from isolated brain mitochondria and indirectly modulates store-operated Ca2+ channels. We suggest that some of the effects of FFA on plasma membrane ion channels could be explained, at least partially, by its ability to modulate the mitochondrial Ca2+ homeostasis.

The mitochondrial ADP/ATP carrier: functional and structural studies in the route of elucidating pathophysiological aspects.

The mitochondrial ADP/ATP carrier plays a central role in aerobic cell energetics by providing to the cytosol the ATP generated by oxidative phosphorylation. Though discovered around 40 years ago owing to the existence of unique inhibitors and in spite of numerous experimental approaches, this carrier, which stands as a model of the mitochondrial solute carriers keeps some long-standing mystery. There are still open challenging questions among them the precise ADP/ATP transport mechanism, the functional oligomeric state of the carrier and relationships between human ADP/ATP carrier dysfunctioning and pathologies. Deciphering the 3D structure of this carrier afforded a considerable progress of the knowledge but requires now additional data focused on molecular dynamics from this static picture. State of the art in this topic is reviewed and debated in this paper in view of better comprehending origin of the discrepancies in these questions and, finally, the multiple physiological roles of this carrier in eukaryotic cell economy.

Structure-function relationships of the C-terminal end of the Saccharomyces cerevisiae ADP/ATP carrier isoform 2.

The adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in the cellular energy metabolism. Two regions of Anc2p from Saccharomyces cerevisiae are specifically photolabeled using a photoactivable ADP derivative; they are the central matrix loop, m2, and the C-terminal end. To get more insights into the structure-function relationships of the C-terminal region during nucleotide transport, we have developed two independent approaches. In the first we have deleted the last eight amino acids of Anc2p (Anc2pDeltaCter) and demonstrated that the C-terminal end of Anc2p plays an essential role in yeast growth on a non-fermentable carbon source. This resulted from impaired nucleotide binding properties of the Anc2pDeltaCter variant in line with conversion of ADP binding sites from high to low affinity. In the second we probed the ligand-induced conformational changes of Anc2p C-terminal end (i) by assessing its accessibility to anti-C-terminal antibodies and (ii) by measuring intrinsic fluorescence changes of an Anc2p mutant containing only one tryptophan residue located at its C-terminal end (Anc2p3Y-u). We show that the C-terminal region is no further accessible to antibodies when Anc2p binds non-transportable analogues of ADP. Besides, Trp-316 fluorescence is highly increased upon ligand binding, suggesting large conformational changes. Taken together, our results highlight the involvement of the Anc2p C-terminal region in nucleotide recognition, binding, and transport.

Conformation-dependent swinging of the matrix loop m2 of the mitochondrial Saccharomyces cerevisiae ADP/ATP carrier.

Structure-function relationships of the membrane-embedded Saccharomyces cerevisiae mitochondrial ADP/ATP carrier were investigated through two independent approaches, namely, limited proteolysis and cysteine labeling. Experiments were carried out in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport that bind to two distinct conformers involved in the translocation process. The proteolysis approach allowed us to demonstrate (i) that N- and C-terminal extremities of ADP/ATP carrier are facing the intermembrane space and (ii) that the central region of the carrier corresponding to the matrix loop m2 is accessible to externally added trypsin in a conformation-sensitive manner, being cleaved at the Lys163-Gly164 and Lys178-Thr179 bonds in the carrier-CATR and the carrier-BA complexes, respectively. The cysteine labeling approach was carried out on the S161C mutant of the ADP/ATP carrier. This variant of the carrier is fully active, displaying nucleotide transport kinetic parameters and inhibitor binding properties similar to that of wild-type carrier. Alkylation experiments, carried out on mitochondria with the nonpermeable reagents eosin-5-maleimide and iodoacetamidyl-3,6-dioxaoctanediamine-biotin, showed that Cys 161 is accessible from the outside in the carrier-CATR complex, whereas it is masked in the carrier-BA complex. Taken together, our results indicate that the matrix loop m2 connecting the transmembrane helices H3 to H4 intrudes to some extent into the inner mitochondrial membrane. Its participation in the translocation of ADP/ATP is strongly suggested, based on the finding that its accessibility to reagents added outside mitochondria is modified according to the conformational state of the carrier.

Functional characterization and purification of a Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein.

A recombinant fusion protein combining the mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p), both from Saccharomyces cerevisiae, has been genetically elaborated with the aim of increasing the polar surface area of the carrier to facilitate its crystallization. The gene encoding the his-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2. The chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, which demonstrated its transport activity. The kinetic exchange properties of Anc2-Cyc1(His6)p and the wild type his-tagged carrier Anc2(His6)p were very similar. However, Anc2-Cyc1(His6)p restored cell growth less efficiently than Anc2(His6)p which correlates with the lower amount found in mitochondria. Purification of Anc2-Cyc1(His6)p in complex with carboxyatractyloside (CATR), a high affinity inhibitor of ADP/ATP transport, was achieved by combining ion-exchange chromatography and ion-metal affinity chromatography in the presence of LAPAO, an aminoxide detergent. As characterized by absorption in the visible range, heme was found to be present in isolated Anc2-Cyc1(His6)p, giving the protein a red color. Large-scale purification of Anc2-Cyc1(His6)p-CATR complex opens up novel possibilities for the use of crystallographic approaches to the yeast ADP/ATP carrier.

Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox. Structural and functional comparison of p40phox and p67phox SH3 domains.

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.

Valine 181 is critical for the nucleotide exchange activity of human mitochondrial ADP/ATP carriers in yeast.

We isolated yeast Saccharomyces cerevisiae cells transformed with one of the three human adenine nucleotide carrier genes (HANC) that exhibited higher growth capacity than previously observed. The HANC genes were isolated from these clones, and we identified two independent mutations of HANC that led to replacement of valine 181 located in the fourth transmembrane segment by methionine or phenylalanine. Tolerance of this position toward substitution with various amino acids was systematically investigated, and since HANC/V181M was among the most efficient in growth complementation, it was more extensively studied. Here we show that increased growth capacities were associated with higher ADP/ATP exchange activities and not with higher human carrier amount in yeast mitochondria. These results are discussed in the light of the bovine Ancp structure, that shares more than 90% amino acid identity with Hancps, and its interaction with the lipid environment.

X-ray spectroscopy and X-ray diffraction at wavelengths near the K-absorption edge of phosphorus.

Phosphorus is an abundant element in living organisms. It is traceable by its X-ray absorption spectrum which shows a strong white line at its K-edge, comparable with that observed for the L(III) edges of rare earth ions. With purple membrane, the variation of the imaginary part of the anomalous dispersion of phosphorus is found to be close to 20 anomalous electron units. Anomalous diffraction experiments at wavelengths near the K-absorption edge of phosphorus confirm this result. The spatial distribution of lipids derived from anomalous diffraction agrees with earlier results from neutron diffraction. Test experiments on single crystals of the carrier protein using 5.76 A photons gave a first low-resolution diffraction pattern. Various techniques of crystal mounting were attempted. In addition, fluorescence measurements on a solution of threonine synthase appear to hint at a change of the phosphate environment of the cofactor upon activator binding.