Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.

Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region.

The transduction of transmembrane electric fields into protein motion has an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSDs) carry out these functions through reorientations of positive charges in the S4 helix. Here, we determined crystal structures of the Ciona intestinalis VSD (Ci-VSD) in putatively active and resting conformations. S4 undergoes an ~5-Å displacement along its main axis, accompanied by an ~60° rotation. This movement is stabilized by an exchange in countercharge partners in helices S1 and S3 that generates an estimated net charge transfer of ~1 eo. Gating charges move relative to a ''hydrophobic gasket' that electrically divides intra- and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent enzymes and ion channels.

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 microM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Abeta1-42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.

The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen/oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS-Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson's disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders.

Febrifugine, the bioactive constituent of one of the 50 fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity, though its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis and inflammatory disease. We recently demonstrated that halofuginone (HF), a widely studied derivative of febrifugine, inhibits the development of T(H)17-driven autoimmunity in a mouse model of multiple sclerosis by activating the amino acid response (AAR) pathway. Here we show that HF binds glutamyl-prolyl-tRNA synthetase (EPRS), inhibiting prolyl-tRNA synthetase activity; this inhibition is reversed by the addition of exogenous proline or EPRS. We further show that inhibition of EPRS underlies the broad bioactivities of this family of natural product derivatives. This work both explains the molecular mechanism of a promising family of therapeutics and highlights the AAR pathway as an important drug target for promoting inflammatory resolution.

The tumor accumulation of nanomedicines relies on the enhanced permeability and retention (EPR) effect. In the last 5-10 years, it has been increasingly recognized that there is a large inter- and intra-individual heterogeneity in EPR-mediated tumor targeting, explaining the heterogeneous outcomes of clinical trials in which nanomedicine formulations have been evaluated. To address this heterogeneity, as in other areas of oncology drug development, we have to move away from a one-size-fits-all tumor targeting approach, towards methods that can be employed to individualize and improve nanomedicine treatments. To this end, efforts have to be invested in better understanding the nature, the complexity and the heterogeneity of the EPR effect, and in establishing systems and strategies to enhance, combine, bypass and image EPR-based tumor targeting. In the present manuscript, we summarize key studies in which these strategies are explored, and we discuss how these approaches can be employed to enhance patient responses.

Inclusion of a tumor-targeting molecule in nanosized delivery systems increases their in vivo efficacy. However, the biodistribution and pharmacokinetics of the uptake of such particles have not yet been well addressed. Several recent papers have suggested that tumor-targeting ligands function primarily to increase intracellular uptake of the nanocomplex and do not influence tumor localization. However, other reports indicate that they do play a role in the accumulation in the tumor. One difference might be the presence or absence of poly-[ethylene glycol] (PEG) in the complex and its impact on the enhanced permeability and retention (EPR) effect. Further studies are clearly needed to more fully elucidate the influence of composition on tumor-targeted, systemic delivery of nanoparticles.

Targeting tumors with long-circulating nano-scaled carriers is a promising strategy for systemic cancer treatment. Compared with free small therapeutic agents, nanocarriers can selectively accumulate in solid tumors through the enhanced permeability and retention (EPR) effect, which is characterized by leaky blood vessels and impaired lymphatic drainage in tumor tissues, and achieve superior therapeutic efficacy, while reducing side effects. In this way, drug-loaded polymeric micelles, i.e. self-assemblies of amphiphilic block copolymers consisting of a hydrophobic core as a drug reservoir and a poly(ethylene glycol) (PEG) hydrophilic shell, have demonstrated outstanding features as tumor-targeted nanocarriers with high translational potential, and several micelle formulations are currently under clinical evaluation. This review summarizes recent efforts in the development of these polymeric micelles and their performance in human studies, as well as our recent progress in polymeric micelles for the delivery of nucleic acids and imaging.

A key question for the understanding of photosynthetic water oxidation is whether the four oxidizing equivalents necessary to oxidize water to dioxygen are accumulated on the four Mn ions of the oxygen-evolving complex (OEC), or whether some ligand-centered oxidations take place before the formation and release of dioxygen during the S(3) ->> [S(4)] ->> S(0) transition. Progress in instrumentation and flash sample preparation allowed us to apply Mn Kbeta X-ray emission spectroscopy (Kbeta XES) to this problem for the first time. The Kbeta XES results, in combination with Mn X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) data obtained from the same set of samples, show that the S(2) ->> S(3) transition, in contrast to the S(0) ->> S(1) and S(1) ->> S(2) transitions, does not involve a Mn-centered oxidation. On the basis of new structural data from the S(3)-state, manganese mu-oxo bridge radical formation is proposed for the S(2) ->> S(3) transition, and three possible mechanisms for the O-O bond formation are presented.

Ion channels catalyze the selective transfer of ions across the membrane in response to a variety of stimuli. These channels gate by controlling the access of ions to a centrally located water-filled pore. The crystal structure of the Streptomyces lividans potassium channel (KcsA) has allowed a molecular exploration of this mechanism. Electron paramagnetic resonance (EPR) studies have uncovered significant conformational changes at the intracellular end of the second transmembrane helix (TM2) upon gating. We have used site-directed spin labeling (SDSL) and EPR spectroscopy in an attempt to quantify the structural rearrangements of the KcsA TM2 bundle underlying the transition from the closed to the open state. Under conditions favoring the closed and open conformations, 10 intersubunit distances were obtained across TM2 segments from tandem dimer constructs. Analysis of these data points to a mechanism in which each TM2 helix tilts away from the permeation pathway, towards the membrane plane, and rotates about its helical axis, supporting a scissoring-type motion with a pivot point near residues 107-108. These movements are accompanied by a large increase in the diameter of the vestibule below the central water-filled cavity.

An efficient noninvasive method for in vivo imaging of tumor oxygenation by using a low-field magnetic resonance scanner and a paramagnetic contrast agent is described. The methodology is based on Overhauser enhanced magnetic resonance imaging (OMRI), a functional imaging technique. OMRI experiments were performed on tumor-bearing mice (squamous cell carcinoma) by i.v. administration of the contrast agent Oxo63 (a highly derivatized triarylmethyl radical) at nontoxic doses in the range of 2-7 mmol/kg either as a bolus or as a continuous infusion. Spatially resolved pO(2) (oxygen concentration) images from OMRI experiments of tumor-bearing mice exhibited heterogeneous oxygenation profiles and revealed regions of hypoxia in tumors (<10 mmHg; 1 mmHg = 133 Pa). Oxygenation of tumors was enhanced on carbogen (95% O(2)/5% CO(2)) inhalation. The pO(2) measurements from OMRI were found to be in agreement with those obtained by independent polarographic measurements using a pO(2) Eppendorf electrode. This work illustrates that anatomically coregistered pO(2) maps of tumors can be readily obtained by combining the good anatomical resolution of water proton-based MRI, and the superior pO(2) sensitivity of EPR. OMRI affords the opportunity to perform noninvasive and repeated pO(2) measurements of the same animal with useful spatial (approximately 1 mm) and temporal (2 min) resolution, making this method a powerful imaging modality for small animal research to understand tumor physiology and potentially for human applications.

A fundamental challenge in the redox biology of Mycobacterium tuberculosis (Mtb) is to understand the mechanisms involved in sensing redox signals such as oxygen (O2), nitric oxide (NO), and nutrient depletion, which are thought to play a crucial role in persistence. Here we show that Mtb WhiB3 responds to the dormancy signals NO and O2 through its iron-sulfur (Fe-S) cluster. To functionally assemble the WhiB3 Fe-S cluster, we identified and characterized the Mtb cysteine desulfurase (IscS; Rv3025c) and developed a native enzymatic reconstitution system for assembling Fe-S clusters in Mtb. EPR and UV-visible spectroscopy analysis of reduced WhiB3 is consistent with a one-electron reduction of EPR silent [4Fe-4S]2+ to EPR visible [4Fe-4S]+. Atmospheric O2 gradually degrades the WhiB3 [4Fe-4S]2+ cluster to generate a [3Fe-4S]+ intermediate. Furthermore, EPR analysis demonstrates that NO forms a protein-bound dinitrosyl-iron-dithiol complex with the Fe-S cluster, indicating that NO specifically targets the WhiB3 Fe-S cluster. Our data suggest that the mechanism of WhiB3 4Fe-4S cluster degradation is similar to that of fumarate nitrate regulator. Importantly, Mtb DeltawhiB3 shows enhanced growth on acetate medium, but a growth defect on media containing glucose, pyruvate, succinate, or fumarate as the sole carbon source. Our results implicate WhiB3 in metabolic switching and in sensing the physiologically relevant host signaling molecules NO and O2 through its [4Fe-4S] cluster. Taken together, our results suggest that WhiB3 is an intracellular redox sensor that integrates environmental redox signals with core intermediary metabolism.

An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mössbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and Mössbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The Mössbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.

Ca(2+)-triggered, synchronized synaptic vesicle fusion underlies interneuronal communication. Complexin is a major binding partner of the SNARE complex, the core fusion machinery at the presynapse. The physiological data on complexin, however, have been at odds with each other, making delineation of its molecular function difficult. Here we report direct observation of two-faceted functions of complexin using the single-vesicle fluorescence fusion assay and EPR. We show that complexin I has two opposing effects on trans-SNARE assembly: inhibition of SNARE complex formation and stabilization of assembled SNARE complexes. Of note, SNARE-mediated fusion is markedly stimulated by complexin, and it is further accelerated by two orders of magnitude in response to an externally applied Ca(2+) wave. We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca(2+) and Ca(2+)-sensing fusion effectors in neurotransmitter release.

The objective of this study was to investigate the molecular weight (MW) and time-dependence of the phenomenon termed "the enhanced permeability and retention" (EPR) effect in solid tumor, in particular to determine and define the early phase accumulation of macromolecules in tumor and normal tissues and the relationship between blood concentration and tissue clearance. As a model, radioiodinated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers of MW ranging from 4.5 K to 800 K were administered i.v. to mice bearing sarcoma 180 tumor. Within 10 min all HPMA copolymers accumulated effectively in the tumor regardless of MW (1.0-1.5% of injected dose per g of tumor). However, higher MW copolymers >> 50 K) showed significantly increased tumor accumulation after 6 h, while the lower MW copolymers (< 40 K) were cleared rapidly from tumor tissue due to rapid diffusion back into the bloodstream. Blood clearance was also MW-dependent; the lower MW copolymers displayed rapid clearance, with kidney radioactivity of the copolymers of MW < 20 K representing 24% of injected dose per g kidney at 1 min after i.v. administration. Within 10 min these copolymers passed through the kidney and were excreted in the urine. Higher MW copolymers consistently showed kidney levels of 3-5% dose per g kidney in the early phase with no time-dependent accumulation in kidney. There was also no progressive accumulation in muscle or liver, regardless of polymer MW. These results suggest the "EPR effect" in solid tumor primarily arises from in the difference in clearance rate between the solid tumor and the normal tissues after initial penetration of the polymers into these tissues.

The amyloid beta-peptide (A beta)-associated free radical oxidative stress model for neuronal death in Alzheimer's disease (AD) brain predicts that neuronal protein oxidation is a consequence of A beta-associated free radicals [8]. In this study we have used both in vitro and in vivo models of beta-amyloid (A beta) toxicity to detect free radical induced oxidative stress by the measure of protein carbonyl levels. These model systems employed cultured hippocampal neurons exposed to exogenous synthetic A beta(1-42) and transgenic Caenorhabditis elegans (C. elegans) animals expressing A beta(1-42). We also investigated the importance of the A beta(1-42) Met35 residue for free radical formation in peptide solution and for peptide-induced protein oxidation and neuronal toxicity in these model systems. A beta(1-42) in solution yielded an EPR spectrum, suggesting that free radicals are associated with this peptide; however, neither the reverse [A beta(42-1)] nor methionine-substituted peptide [A beta(1-42)Met35Nlc] gave significant EPR spectra, suggesting the importance of the methionine residue in free radical formation. A beta(1-42) addition to cultured hippocampal neurons led to both neurotoxicity (30.1% cell death, p < 0.001) and increased protein oxidation (158% of controls, p < 0.001). and both of those effects were not observed with reverse or Met35Nle substituted peptides. C. elegans transgenic animals expressing human A beta(1-42) also had significantly increased in vivo protein carbonyls (176% of control animals, p < 0.001), consistent with our model. In contrast, transgenic animals with a Met35cys substitution in A beta(1-42) showed no increased protein carbonyls in vivo, in support of the hypothesis that methionine is important in A beta-associated free radical oxidative stress. These results are discussed with reference to the A beta-associated free radical oxidative stress model of neurotoxicity in AD brain.

Targeted drug delivery to tumor sites is one of the ultimate goals in drug delivery. Recent progress in nanoparticle engineering has certainly improved drug targeting, but the results are not as good as expected. This is largely due to the fact that nanoparticles, regardless of how advanced they are, find the target as a result of blood circulation, like the conventional drug delivery systems do. Currently, the nanoparticle-based drug delivery to the target tumor tissues is based on wrong assumptions that most of the nanoparticles, either PEGylated or not, reach the target by the enhanced permeation and retention (EPR) effect. Studies have shown that so-called targeting moieties, i.e., antibodies or ligands, on the nanoparticle surface do not really improve delivery to target tumors. Targeted drug delivery to tumor sites is associated with highly complex biological, mechanical, chemical and transport phenomena, of which characteristics vary spatiotemporally. Yet, most of the efforts have been focused on design and surface manipulation of the drug carrying nanoparticles with relatively little attention to other aspects. This article examines the current misunderstandings and the main difficulties in targeted drug delivery.

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels. Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here, we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the three-dimensional structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active center featuring the 'histidine brace' active site, but is distinct in terms of its active site details and its EPR spectroscopy. The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs.

In the present perspective, we give a critical review on the coordination chemistry of the metal ions copper and zinc to the amyloid-beta (Abeta) peptide; such complexes have been linked to Alzheimer's disease. We focus on two main issues: the identification of the coordination sphere of the Cu(ii) and Zn(ii) ions and the affinity of these metal ions towards the peptide. With the aim to come up with as few as possible valuable structural models and binding affinity values, we critically review the divergent propositions reported in the literature and take into account the experimental differences and the limits of the methods used in the published studies. We propose that: (i) the conditional dissociation constant of the Cu(Abeta) complexes lies in the range of 10 pM to 100 nM, with a preference for the region between 100 pM to 1 nM. (ii) Two most likely coordination modes for the predominant form of the Cu(Abeta) complexes at physiological pH can be retained, both being 3N1O distorted square planar. In the first model, the Cu(ii) ion is coordinated by the Ntau atoms of the three His residues and the carboxylate of the Asp1. In the second model, both the N-terminus and the carboxylate functions of Asp1 are ligated together with the Ntau of His6 and of His13 (or His14). An equilibrium between these two forms at room temperature, and a preferentially freezing out of the second one would explain most of the divergences in the published results (in particular, between those obtained by EPR and NMR). (iii) The apparent dissociation constants of Zn(Abeta) in various buffers are in the range of 1 to 20 muM (a 10 times lower conditional dissociation constant can be estimated. (iv) For the Zn(ii) coordination, the implication of the three His and the Asp1 residues is consensual. The Asp1 can be coordinated by the carboxylate and/or the N-terminus functions. Additional ligands are possible, such as Glu11 or H(2)O.

Parameters of relevance to oximetry with Overhauser magnetic resonance imaging (OMRI) have been measured for three single electron contrast agents of the triphenylmethyl type. The single electron contrast agents are stable and water soluble. Magnetic resonance properties of the agents have been examined with electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and dynamic nuclear polarization (DNP) at 9.5 mT in water, isotonic saline, plasma, and blood at 23 and 37 degreesC. The relaxivities of the agents are about 0.2-0.4 mM-1s-1 and the DNP enhancements extrapolate close to the dipolar limit. The agents have a single, narrow EPR line, which is analyzed as a Voigt function. The linewidth is measured as a function of the agent concentration and the oxygen concentration. The concentration broadenings are about 1-3 microT/mM and the Lorentzian linewidths at infinite dilution are less than 1 microT in water at room temperature. The longitudinal electron spin relaxation rate is calculated from the DNP enhancement curves. The oxygen broadening in water is about 50 microT/mM O2 at 37 degreesC. These agents have good properties for oximetry with OMRI.

The structure of the nitrogenase MoFe-protein from Azotobacter vinelandii has been refined to 2.0 A resolution in two oxidation states. EPR studies on the crystals indicate that the structures correspond to the spectroscopically assigned oxidized (P(OX)/M(OX)) and the native or dithionite-reduced (P(N)/M(N)) forms of the enzyme. Both MoFe-protein structures are essentially identical, with the exception of the P-cluster. The MoFe-protein P-cluster in each state is found to contain eight Fe and seven S atoms. Interconversion between the two redox states involves movement of two Fe atoms and an exchange of protein coordination for ligands supplied by a central S atom. In the oxidized P(OX) state, the cluster is coordinated by the protein through six cysteine ligands, Ser-beta188 O gamma, and the backbone amide of Cys-alpha88. In the native P(N) state, Ser-beta188 O gamma and the amide N of Cys-alpha88 no longer coordinate the cluster due to movement of their coordinated Fe atoms toward the central sulfur. Consequently, this central sulfur adopts a distorted octahedral environment with six surrounding Fe atoms. A previously described model of the P-cluster containing 8Fe-8S likely reflects the inappropriate modeling of a single structure to a mixture of these two P-cluster redox states. These observed redox-mediated structural changes of the P-cluster suggest a role for this cluster in coupling electron transfer and proton transfer in nitrogenase.

A vaccine capable of stimulating protective antiviral antibody responses is needed to curtail the global AIDS epidemic caused by HIV-1. Although rarely elicited during the course of natural infection or upon conventional vaccination, the membrane-proximal ectodomain region (MPER) of the HIV-1 glycoprotein of M(r) 41,000 (gp41) envelope protein subunit is the target of 3 such human broadly neutralizing antibodies (BNAbs): 4E10, 2F5, and Z13e1. How these BNAbs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but such information might offer important insight into a worldwide health imperative. Here, EPR and NMR techniques were used to define the manner in which these BNAbs differentially recognize viral membrane-encrypted residues configured within the L-shaped helix-hinge-helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identified. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. In contrast, Z13e1 effects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, BNAbs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features important for targeted vaccine design have been revealed, the implications of which extend to BNAb targets on other viral fusion proteins.

The newly introduced sulfhydryl reductant tris(2-carboxyethyl)phosphine (TCEP) is a potentially attractive alternative to commonly used dithiothreitol (DTT). We compare properties of DTT and TCEP important in protein biochemistry, using the motor enzyme myosin as an example protein. The reductants equally preserve myosin's enzymatic activity, which is sensitive to sulfhydryl oxidation. When labeling with extrinsic probes, DTT inhibits maleimide attachment to myosin and must be removed before labeling. In contrast, maleimide attachment to myosin was achieved in the presence of TCEP, although with less efficiency than no reductant. Surprisingly, iodoacetamide attachment to myosin was nearly unaffected by either reductant at low (0.1 mM) concentrations. In electron paramagnetic resonance (EPR) spectroscopy utilizing nitroxide spin labels, TCEP is highly advantageous: spin labels are two to four times more stable in TCEP than DTT, thereby alleviating a long-standing problem in EPR. During protein purification, Ni(2+) concentrations contaminating proteins eluted from Ni(2+) affinity columns cause rapid oxidation of DTT without affecting TCEP. For long-term storage of proteins, TCEP is significantly more stable than DTT without metal chelates such as EGTA in the buffer, whereas DTT is more stable if metal chelates are present. Thus TCEP has advantages over DTT, although the choice of reductant is application specific.