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PubMed1996

Photophysics of Supercomplexes. Adduct between Ru(bpy)(CN)(4)(2-) and the [32]ane-N(8)H(8)(8+) Polyaza Macrocycle.

The formation of a supercomplex between the Ru(bpy)(CN)(4)(2-) (bpy = 2,2'-bipyridine) complex and the [32]ane-N(8)H(8)(8+) macrocycle (1) has been studied in water and in acetonitrile. In acetonitrile, supercomplex formation is accompanied by (i) large hypsochromic shifts in the absorption spectrum (color changes from deep violet to yellow) and in the emission spectrum, (ii) large anodic shifts in standard oxidation (0.73 V) and reduction (0.37 V) potentials, (iii) typical shifts of (1)H-NMR signals for the macrocycle N-bound protons and the complex bipyridine protons, and (iv) a large increase in the MLCT excited-state lifetime of the complex. In water, the spectral shifts and the changes in standard potential are much less pronounced, but supercomplex formation is evidenced by (13)C-NMR (and (1)H-NMR) and by emission lifetime changes. In both solvents, supercomplex formation is complete in 1:1, 1.0 x 10(-4) M solutions, indicating very large stability constant values. A structure of the supercomplex with the macrocycle bound in a "boat" conformation to the four cyanide ligands of the complex, plausible in terms of molecular models, is consistent with all the experimental data. In water, the supercomplex further associates with added negative species containing carboxylate functions, as shown by partial reversal of the lifetime changes. When the added species is also a potential electron transfer quencher (such as, e.g., Rh(dcb)(3)(3-), dcb = 4,4'-dicarboxy-2,2'-bipyridine), however, association is not accompanied by quenching. This behavior is attributed to the structure of the supercomplex-quencher adduct, in which the macrocycle acts as an insulating spacer between the excited complex and the quencher.

Journal Article↗
PubMed2005

Ribosome exit tunnel can entropically stabilize alpha-helices.

Several experiments have suggested that newly synthesized polypeptide chains can adopt helical structures deep within the ribosome exit tunnel. We hypothesize that confinement in the roughly cylindrical tunnel can entropically stabilize alpha-helices. The hypothesis is validated by using theory and simulations of coarse-grained off-lattice models. The model helix, which is unstable in the bulk, is stabilized in a cylindrical cavity provided the diameter (D) of the cylinder exceeds a critical value D*. When D < D* both the helical content and the helix-coil transition temperature (T(f)) decrease abruptly. Surprisingly, we find that the stability of the alpha-helix depends on the number (N) of amino acid residues. Entropic stabilization, as measured by changes in T(f), increases nonlinearly as N increases. The simulation results are in quantitative agreement with a standard helix-coil theory that takes into account entropy cost of confining a polypeptide chain in a cylinder. The results of this work are in qualitative accord with most of the findings of a recent experiment in which N-dependent ribosome-induced helix stabilization of transmembrane sequences was measured by fluorescence resonance energy transfer.

Computer Simulation↗
PubMed2006

Discovery of Helicobacter pylori shikimate kinase inhibitors: bioassay and molecular modeling.

Shikimate kinase (SK) is the fifth enzyme in the shikimate pathway and catalyzes the phosphate transfer from ATP to shikimate in generating shikimate 3-phosphate and ADP. SK has been developed as a promising target for the discovery of antibacterial agents. In this report, two small molecular inhibitors (compound 1, 3-methoxy-4-{[2-({2-methoxy-4-[(4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]phenoxy}methyl)benzyl]oxy}benzaldehyde; compound 2, 5-bromo-2-(5-{[1-(3,4-dichlorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}-2-furyl)benzoic acid) against Helicobacter pylori SK (HpSK) were successfully identified with IC(50) values of 5.5+/-1.2 and 6.4+/-0.4 microM, respectively. The inhibition kinetics shows that compound 1 is a noncompetitive inhibitor with respect to both shikimate and MgATP, and compound 2 is a competitive inhibitor toward shikimate and noncompetitive inhibitor with respect to MgATP. The surface plasmon resonance (SPR) technology based analysis reveals that the equilibrium dissociation constants (K(D)s) of compounds 1 and 2 with HpSK enzyme are 4.39 and 3.74 microM, respectively. The molecular modeling and docking of two inhibitors with HpSK reveals that the active site of HpSK is rather roomy and deep, forming an L-shape channel on the surface of the protein, and compound 1 prefers the corner area of L-shape channel, while compound 2 binds the short arm of the channel of SK in the binding interactions. It is expected that our current work might supply useful information for the development of novel SK inhibitors.

Algorithms↗
PubMed1991

Dynamic rehabilitation of the paralyzed face: III: Balanced coupling of oral and ocular musculature from the intact side in the canine.

We have recently demonstrated the feasibility of artificial agonist/antagonist coupling between intact facial and reinnervated strap muscles in the rabbit. The present study broadens this principle to involve bi-level cross-facial innervation. After severing the facial innervation unilaterally in four dogs, a nerve pedicle from a cervical motor nerve was implanted into the orbicularis oris and from the deep temporal nerve into the orbicularis oculi. After neurotization (5.6 months), the pedicles were electrically stimulated to verify muscular response. Graded contraction on the intact side was induced by stimulating the ipsilateral facial nerve with currents of various pulse widths. The resulting compression of a strain gauge on the intact face triggered a two-channel, opto-isolated, pulse width-modulated stimulator to produce agonistic graded contraction at one level of the reinnervated side (e.g., oral) and reciprocal relaxation in its reinnervated counterpart (e.g., ocular). The sophistication of the present model--as compared to the original pilot study--brings us one step closer to dynamic human facial rehabilitation.

Animals↗
PubMed2002

Adeno-associated virus-mediated transfer of human acid maltase gene results in a transient reduction of glycogen accumulation in muscle of Japanese quail with acid maltase deficiency.

Glycogen storage disease type II (GSD II), Pompe's disease, is caused by the deficiency of acid alpha-D-glucosidase (GAA) in lysosome and is the most common form of GSD in Taiwan. Most cases are the infantile form. The disease is relentless and most patients die of cardiac failure and respiratory tract infection in the first year of life. At present, no treatment has been proved effective for this fatal disease. The applicability of enzyme replacement therapy is under investigation. However, high price and transient efficiency are the major problems to be solved. Accordingly, gene therapy by viral method has been conducted. In this study we constructed a plasmid that contained 5'-shortened BglII-NotI fragment human GAA cDNA, downstream of CMV promoter and bovine growth hormone polyadenylation signal, as well as AAV ITR region. When fibroblasts obtained from GSD II patients were cultured and infected with rAAV-GAA, the GAA activity of the fibroblasts increased four- to five-fold. Using acid maltase deficient (AMD) Japanese quail as the animal model, rcAAV-GAA 0.1 ml per site (1 x 10(9)-10) particles), totally 10 different sites to make 1 ml (1 x 10(1)0-11) particles), was injected into unilateral deep pectoral muscle of AMD quails. Medium (hepes) was only injected in the same way into the contralateral deep pectoral muscle to serve as control. Four days after injection, PAS staining showed disappearance of the glycogenosomes with regeneration of myocytes surrounding the intramuscular injected area as compared with the contralateral muscle of the same birds. Using anti-GAA monoclonal antibody, GAA was demonstrated on the regenerated myocytes by immunohistochemical staining and absent on the contralateral muscle of the same birds. Nevertheless, T lymphocytes infiltration was noted in both the rcAAV-GAA and hepes (medium) injected muscles and more prominent in the rcAAV-GAA-injected site. Functional evaluation demonstrated that wing flapping movement improved with wide flapping in the rAAV-GAA injected side, but not in the counterpart. Unfortunately, these histochemical and functional improvements faded away in 14 days, probably due to destruction of rcAAV by cell-mediated immunity of infiltrated T cells. Taken together, the present study suggests that rAAV can enter either human or quail cells and express and effectively reduce the glycogen accumulation in the skeletal muscle of AMD quails. These preliminary results are similar to these of low-dose rGAA replacement therapy. The mechanisms underlying the induction of cell-mediated immunity are unknown. How to elevate the number of packaged AAV, enhance the infectivity of AAV and reduce cell-mediated immunity must be solved in the future.

Animals↗
PubMed2001

Structural rationale for the modulation of abscess formation by Staphylococcus aureus capsular polysaccharides.

Staphylococcus aureus is a medically important bacterial pathogen that is a common cause of superficial and deep-seated abscesses in humans. Most S. aureus isolates produce either a serotype 5 or 8 capsular polysaccharide (CP) that has been shown to enhance bacterial virulence. We investigated the role of S. aureus CPs in modulating abscess formation in an experimental animal model of intraabdominal infection. Structural studies of CP8 revealed that it has a zwitterionic charge motif conferred by the negatively charged carboxyl group of N-acetylmannosaminuronic acid and free amino groups available on partially N-acetylated fucosamine residues. We report that purified CP5 and CP8 facilitated intraabdominal abscess formation in animals when given i.p. with a sterile cecal contents adjuvant. Chemical modifications that neutralized the positively or negatively charged groups on CP8 abrogated its ability to provoke abscesses. Rats prophylactically treated with CP8 s.c. were protected against abscess formation induced by homologous or heterologous zwitterionic polysaccharides. Likewise, treatment with CP8 protected against challenge with viable S. aureus strains PS80 (a capsule type 8 strain) or COL (a methicillin-resistant capsule type 5 strain). Purified CP8 was a potent activator of rat and human CD4(+) T cells in vitro. When transferred to naive rats, these activated T cells modulated the development of intraabdominal abscess formation. These results provide a structure/function rationale for abscess formation by S. aureus and expand the sphere of encapsulated organisms that interact directly with T cells to regulate this host response to bacterial infection.

Abdominal Abscess↗
PubMed2001

Approach and issues relating to shield material design to protect astronauts from space radiation.

One major obstacle to human space exploration is the possible limitations imposed by the adverse effects of long-term exposure to the space environment. Even before human spaceflight began, the potentially brief exposure of astronauts to the very intense random solar energetic particle (SEP) events was of great concern. A new challenge appears in deep space exploration from exposure to the low-intensity heavy-ion flux of the galactic cosmic rays (GCR) since the missions are of long duration and the accumulated exposures can be high. Since aluminum (traditionally used in spacecraft to avoid potential radiation risks) leads to prohibitively expensive mission launch costs, alternative materials need to be explored. An overview of the materials related issues and their impact on human space exploration will be given.

Aluminum↗
PubMed1997

Mechanisms of action of the bactericidal/permeability-increasing protein BPI on endotoxin and phospholipid monolayers and aggregates.

We have investigated the mechanisms of interaction of the recombinant N-terminal portion of bactericidal/permeability-increasing protein, rBPI21, with lipopolysaccharide (LPS) isolated from enterobacterial deep rough mutant strains. Experimentally, the ability of rBPI21 to form monolayers at the air/water interface and its action on lipid monolayers were analyzed. We have further studied the interaction of rBPI21 with aggregates from phospholipids and Re mutant LPS by infrared and resonance energy transfer spectroscopy and laser Doppler velocimetry. From monolayer experiments, the molecular area of a single rBPI21 molecule was estimated to be about 12 nm2. At lateral pressures of </=25 mN/m, rBPI21 incorporated into monolayers from negatively charged LPS and phosphatidylglycerol (PG) but not into those from neutral phosphatidylcholine. rBPI21 incorporated not only into monolayers but also into liposomes made from or containing negatively charged phospholipids, reducing the absolute value of the zeta-potential of LPS and PG aggregates. Furthermore, due to intercalation, rBPI21 caused the rigidification of the acyl chains of lipids in the gel as well as in the fluid phase and significantly immobilized their phosphate groups. High concentrations of Mg2+ ions were found to have a protective effect against the action of rBPI21. On the basis of these results, the biophysical characteristics of rBPI21 are discussed and a model is proposed as to how the rBPI21-induced influence on lipid monolayers and bilayers could explain rBPI21-mediated effects on the bacterial membrane.

Anti-Infective Agents↗
PubMed2005

Simulation of medical electron linac bremsstrahlung beam transport in typical shielding materials.

The Monte Carlo N-particle code MCNP version 4C3 was used to investigate the backscattering and transmission of high-energy photons in concrete, iron and lead at deep penetration. A typical bremsstrahlung beam from a 24 MV linac was used, and the transmission up to 15 mean-free paths was studied. Broad beam slab geometry was used. Estimates of the transmission in terms of absorbed dose to tissue ratio and air kerma ratio were performed for the primary and secondary components of the transmitted beam in the three materials. The tissue dose and air kerma buildup factors were calculated and fitted to Berger's equation. Finally, the differential dose albedo values for common reflected angles were determined.

Computer Simulation↗
PubMed2025

A single-cell transcriptomic atlas of the pigtail macaque placenta in late gestation.

The placenta is a complex organ with multiple immune and non-immune cell types that promote fetal tolerance and facilitate the transfer of nutrients and oxygen. The nonhuman primate (NHP) is a key experimental model for studying human pregnancy complications, in part due to similarities in placental structure, which makes it essential to understand how single-cell populations compare across the human and NHP maternal-fetal interface. We constructed a single-cell RNA-Seq (scRNA-Seq) atlas of the placenta from the pigtail macaque ( Macaca nemestrina ) in the third trimester, comprising three different tissues at the maternal-fetal interface: the chorionic villi (placental disc), chorioamniotic membranes, and the maternal decidua. Each tissue was separately dissociated into single cells and processed through the 10X Genomics and Seurat pipeline, followed by aggregation, unsupervised clustering, and cluster annotation. Next, we determined the maternal-fetal origins of cell populations and analyzed single-cell RNA trajectory, Gene Ontology enrichment, and cell-cell communication. Single-cell populations in the pigtail macaque were strikingly similar in their identity and frequency to those found in the human placenta, including cells from trophoblast, stromal cell, immune, and macrophage lineages. An advantage of our approach was the deep sequencing of three tissues at the maternal-fetal interface, which yielded a rich diversity of common and rare single-cell populations. The third-trimester pigtail macaque single-cell atlas enables the identification of cellular subclusters analogous to those in humans and provides a powerful resource for understanding experimental perturbations on the NHP placenta.

Journal Article↗
PubMed2003

Enhanced mantle-to-crust rhenium transfer in undegassed arc magmas.

Variations in the 187Os/188Os isotopic signature of mantle and mantle-derived rocks have been thought to provide a powerful chemical tracer of deep Earth structure. Many studies have inferred from such data that a long-lived, high-rhenium component exists in the deep mantle (187Re is the parent isotope decaying to 187Os, with a half-life of approximately 42 billion years), and that this reservoir probably consists of subducted oceanic crust. The interpretation of these isotopic signatures is, however, dependent on accurate estimates of rhenium and osmium concentrations in all of the main geochemical reservoirs, and the crust has generally been considered to be a minor contributor to such global budgets. In contrast, we here present observations of high rhenium concentrations and low Yb/Re ratios in arc-type melt inclusions. These results indicate strong enrichment of rhenium in undegassed arc rocks, and consequently the continental crust, which results in a crustal estimate of 2 p.p.b. rhenium, as compared to previous estimates of 0.4-0.2 p.p.b. (refs 4, 5). Previous determinations of rhenium in arc materials, which were largely measured on subaerially erupted samples, are likely to be in error owing to rhenium loss during degassing. High mantle-to-crust rhenium fluxes, as observed here, require a revaluation of geochemical models based on the 187Re-187Os decay system.

Journal Article↗
PubMed2000

Head-related transfer functions of the Rhesus monkey.

Head-related transfer functions (HRTFs) are direction-specific acoustic filters formed by the head, the pinnae and the ear canals. They can be used to assess acoustical cues available for sound localization and to construct virtual auditory environments. We measured the HRTFs of three anesthetized Rhesus monkeys (Macaca mulatta) from 591 locations in the frontal hemisphere ranging from -90 degrees (left) to 90 degrees (right) in azimuth and -60 degrees (down) to 90 degrees (up) in elevation for frequencies between 0.5 and 15 kHz. Acoustic validation of the HRTFs shows good agreement between free field and virtual sound sources. Monaural spectra exhibit deep notches at frequencies above 9 kHz, providing putative cues for elevation discrimination. Interaural level differences (ILDs) and interaural time differences (ITDs) generally vary monotonically with azimuth between 0.5 and 8 kHz, suggesting that these two cues can be used to discriminate azimuthal position. Comparison with published subsets of HRTFs from squirrel monkeys (Saimiri sciureus) shows good agreement. Comparison with published human HRTFs from the frontal hemisphere demonstrates overall similarity in the patterns of ILD and ITD, suggesting that the Rhesus monkey is a good acoustic model for these two sound localization cues in humans. Finally, the measured ITDs in the horizontal plane agree well between -40 degrees and 40 degrees in azimuth with those calculated from a spherical head model with a radius of 52 mm, one-half the interaural distance of the monkey.

Animals↗
PubMed2000

The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases.

Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 A) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta-glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.

Amino Acid Sequence↗
PubMed2000

Patellar cartilage deformation in vivo after static versus dynamic loading.

The objective of this study was to test the hypothesis that static loading (squatting at a 90 degrees angle) and dynamic loading (30 deep knee bends) cause different extents and patterns of patellar cartilage deformation in vivo. The two activities were selected because they imply different types of joint loading and reflect a realistic and appropriate range of strenuous activity. Twelve healthy volunteers were examined and the volume and thickness of the patellar cartilage determined before and from 90 to 320s after loading, using a water excitation gradient echo MR sequence and a three-dimensional (3D) distance transformation algorithm. Following knee bends, we observed a residual reduction of the patellar cartilage volume (-5.9+/-2.1%; p<0.01) and of the maximal cartilage thickness (-2.8+/-2.6%), the maximal deformation occurring in the superior lateral and the medial patellar facet. Following squatting, the change of patellar cartilage volume was -4.7+/-1.6% (p<0.01) and that of the maximal cartilage thickness -4.9+/-1.4% (p<0.01), the maximal deformation being recorded in the central aspect of the lateral patellar facet. The volume changes were significantly lower after squatting than after knee bends (p<0.05), but the maximal thickness changes higher (p<0.05). The results obtained in this study can serve to validate computer models of joint load transfer, to guide experiments on the mechanical regulation of chondrocyte biosynthesis, and to estimate the magnitude of deformation to be encountered by tissue-engineered cartilage within its target environment.

Adult↗
PubMed1984

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages.

Endocytosis of immunoglobulin G (IgG)-coated colloidal gold particles in cultured mouse peritoneal macrophages was studied by scanning and transmission electron microscopy. At 4 degrees C, the tracers adhered to the plasma membrane and accumulated in coated pits located in the bottom of furrows or deep invaginations on the cell surface. In the presence of an excess of unlabeled mouse IgG, cellular binding of the tracer was reduced by 80 to 90%. After warming to 37 degrees C, surface-bound tracer particles were rapidly ingested and transported to small and large vesicles lacking membrane coat. From here, they were then passed over to multivesicular bodies and lysosomes characterized by their content of myelin-like figures and other inclusions. Double-labeling experiments with IgG-coated colloidal gold particles of two different sizes (20 and 5 nm diameter) indicated that the plasma membrane was depleted of binding sites after uptake of a polyvalent ligand. The restoration of the binding capacity was a slow process requiring ongoing protein synthesis. On the basis of these observations, a model for endocytosis of immune complexes in macrophages is presented. It includes the following four steps: IgG-containing macromolecular aggregates bind to specific receptors in the plasma membrane. These appear to be preclustered in coated pits or able to move laterally within the membrane even at 4 degrees C. The receptor-ligand complexes are internalized and transferred sequentially to larger uncoated vesicles or endosomes, multivesicular bodies, and lysosomes with inclusions of varying appearance. Receptors and ligands are degraded within the lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Probing the properties of lipopolysaccharide monolayers and their interaction with the antimicrobial peptide polymyxin B by atomic force microscopy.

In contrast to the majority of all known cell types, Gram-negative bacteria have a second membrane, the outer membrane, which is an asymmetric bilayer composed of a phospholipid inner leaflet and a glycolipid outer leaflet. The glycolipid layer, in most cases being composed of a lipopolysaccharide (LPS), is the first target for antimicrobial agents. To get a basic understanding of the membrane-forming properties of LPS, we reconstituted monolayers of deep rough mutant LPS from Salmonella enterica serova Minnesota (R595 LPS), its lipid A moiety, and of the synthetic tetraacyl compound 406 (resembling the biosynthetic lipid A precursor IVa) at the air-water interface of a film balance. The liquid-expanded (LE) and liquid-condensed (LC) domains in the coexisting region were investigated with epifluorescence and, after transferring the monolayer onto mica, as a Langmuir-Blodgett film, with atomic force microscopy (AFM). The fluorescence and the AFM images showed identical domain structure. The higher resolution of the AFM images, however, contained more topographic details. Different heights and adhesion forces between the LE and LC domains could be observed. Differences in the adhesion forces between the AFM tip and the sample were determined in the repulsive and the attractive dynamic scanning modes, demonstrating the importance of a careful interpretation of height images. We propose that an increase in the lateral pressure causing the LE-LC transition of the monolayers leads to a reorientation of the molecules due to a tilt angle between the alkyl chains and the diglucosamine backbone. LPS monolayers have been utilized as a simplified reconstitution model of the outer membrane to study the interaction with antimicrobial agents. We investigated the action of the polycationic peptide polymyxin B (PMB) and found dramatic influences on the domain structures.

Anti-Bacterial Agents↗
PubMed2006

Direct visualization of saposin remodelling of lipid bilayers.

Saposins A, B, C and D are soluble, non-enzymatic proteins that interact with lysosomal membranes to activate the breakdown and transfer of glycosphingolipids. The mechanisms of hydrolase activation and lipid transfer by saposins remain unknown. We have used in situ atomic force microscopy (AFM) with simultaneous confocal fluorescence microscopy to investigate the interactions of saposins with lipid membranes. AFM images of the effect of saposins A, B and C on supported lipid bilayers showed a time and concentration-dependent nucleated spread of membrane transformation. Saposin B produced deep gaps that ultimately filled with granular material, while saposins A and C lead to localized areas of membrane that were reduced in height by approximately 1.5 nm. Fluorescence-labeled saposin C co-localized with the transformed areas of the bilayer, indicating stable binding to the membrane. Fluorescence resonance energy transfer confirmed a direct interaction between saposin C and lipid. Under certain conditions of membrane lipid composition and saposin concentration, extensive bilayer lipid removal was observed. We propose a multi-step mechanism that integrates the structural features and amphipathic properties of the saposin proteins.

Fluorescence Resonance Energy Transfer↗
PubMed1997

Tertiary structural changes and iron release from human serum transferrin.

Iron release from human serum transferrin was investigated by comparison of the extent of bound iron, measured by charge transfer absorption band intensity (465 nm), with changes observed by small-angle solution X-ray scattering (SAXS) for a series of equilibrated samples between pH 5.69 and 7.77. The phosphate buffers used in this study promote iron release at relatively high pH values, with an empirical pK of 6.9 for the convolved release from the two sites. The spectral data reveal that the N-lobe release is nearly complete by pH 7.0, while the C-lobe remains primarily metal-laden. Conversely, the radius of gyration, Rg, determined from the SAXS data remains constant between pH 7.77 and 7.05, and the evolution of Rg between its value observed for the diferric protein at pH 7.77 (31.2+/-0.2 A) and that of the apo protein at pH 5.69 (33.9+/-0.4 A) exhibits an empirical pK of 6.6. While Rg is effectively constant in the pH range associated with iron release from the N-lobe, the radius of gyration of cross-section, Rc, increases from 16.9+/-0.2 A to 17.6+/-0.2 A. Model simulations suggest that two different rotations of the NII domain relative to the NI domain about a hinge deep in the iron-binding cleft of the N-lobe, one parallel with and one perpendicular to the plane of the iron-binding site, can be significantly advanced relative to their holo protein positions while yielding constant Rg and increased Rc values consistent with the scattering data. Rotation of the CII domain parallel with the C-lobe iron-binding site plane can partially account for the increased Rg values measured at low pH; however, no reasonable combined repositioning of the NII and CII domains yields the experimentally observed increase in Rg.

Animals↗