The majority of the Earth's microbial biomass is found in high pressure environments, raising the question of how protein sequences adapt to such environments. Pressure adaptation is more complex, and less well-understood, than adaptation to extreme temperatures since in high pressure environments, these two thermodynamic parameters are often coupled, as in the cold deep-sea or at hydrothermal vents. To begin to address this question, we investigated the functional and folding properties of an exonuclease, Cnase, from the first Gram positive piezophile to be sequenced, Carnobacterium sp. AT7, isolated at 2500 m depth and at ∼2 °C in the Aleutian trench in the North Pacific. We find that Cnase is a bonafide exonuclease, despite its high negative charge. We also find that Cnase largely conserves the structure and folding mechanism of its mesophilic and well-studied homolog, staphylococcal nuclease, Snase, despite significant differences in their sequences.

By performing a comprehensive analysis of nanaerobic respiration in Bacteroides fragilis , we show that this organism maintains capabilities for anaerobic respiration on fumarate and nanaerobic respiration on oxygen simultaneously. The contribution of the two NADH:quinone oxidoreductases and the composition of the quinone pool are the same under both conditions.

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolatedVibrio choleraeNa(+)-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp(49)and Asp(52)(to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR.

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action.

The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae. The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [125I]PAD-1 and [125I]PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32–Met-39 and Phe-131–Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43–Lys-54 and Trp-23–Gly-89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap one another. Unexpectedly, although the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. G140A and E144C), the binding reactivities of [125I]PAD-1 and [125I]PAD-2 to the mutated enzymes were unchanged compared with those of the wild-type enzyme. We also found that photoaffinity labeling by [125I]PAD-1 and [125I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na+-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.

This chapter provides an updated overview of ethical issues and decision making in clinical neuropsychology. It reviews ethical, legal, and professional resources, presents an ethical decision-making model, and emphasizes the importance of a personal commitment to ethical practice. Neuropsychologists practicing in the United States are aware of the American Psychological Association (APA) ethics code and likely turn to the code first when confronting ethical questions or conflicts. Familiarity with ethical and legal resources is usually necessary for addressing complex ethics questions. The information must be organized and applied in a manner that promotes sound decision making, with consideration of personal and situational factors and the dynamic nature of any complex decision making process. As the practice of clinical neuropsychology continues to evolve, the application of professional ethics to clinical practice will need to keep pace. Maintaining open discussion of ethical questions, challenges, and solutions, and sharing experiences with colleagues, will promote ethical practice and be particularly valuable during times of transition.

Containing 50 chapters by some of the most prominent clinical neuropsychologists, the Textbook of Clinical Neuropsychology sets a new standard in the field in its scope, breadth, and scholarship. Unlike most other books in neuropsychology, the Textbook is organized primarily around syndromes, disorders, and related clinical phenomena. Written for the clinician at all levels of training, from the beginner to the journeyman, the Textbook presents contemporary clinical neuropsychology in a comprehensive volume. Chapters are rich with reviews of the literature and clinical case material spanning a range from pediatric to adult and geriatric disorders. Chapter authors are among the most respected in their field, leaders of American Neuropsychology, known for their scholarship and professional leadership. Rarely have so many distinguished members of one discipline been in one volume. This is essential reading for students of neuropsychology, and all others preparing for careers in the field.

Small angle X-ray scattering (SAXS), dynamic light scattering (DLS), and high performance liquid chromatography (HPLC) experiments are performed to support that the inter-micellar distance of Pluronic cubic structures in aqueous solutions is governed by the poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblock copolymer concentration (not the overall polymer concentration) in the solutions. The “as-received (AR)” and “purified (Pure)” F108 solutions show a separate concentration dependence of body-centered cubic (BCC) lattice spacing, when the overall polymer concentration is used as a micellar packing parameter in aqueous solution. When the 22 wt% of non-micellizable polymeric impurities in the AR Pluronic F108 is taken into account, however, a universal concentration dependence of the BCC lattice spacing is observed, unifying results from both AR and Pure F108 solutions. When the PEO-PPO-PEO triblock copolymer concentration from the HPLC analysis is employed as an effective polymer concentration parameter, the universal relationship is observed to provide strong evidence that the polymeric impurities in AR F108 locate themselves in the less dense parts of the interstitial regions on the BCC lattice points, where were occupied by the triblock copolymer micelles. Although the polymeric impurities in AR F108 do not affect the actual triblock concentration dependence of the lattice spacing, they do shift the onset concentration of BCC micellar ordering. In the Pure F108, the onset of BCC packing occurs at the point where the nearest-neighbor radius ( R nn ) in the BCC lattice is approximately equal to the hydrodynamic radius ( R h ), indicating that lattice formation begins upon “hydrodynamic contact” between micelles. In the AR F108, the onset of packing occurs when R nn / R h is approximately 0.9, indicating that, in the presence of the polymeric impurities, micelles must be forced together beyond the point of hydrodynamic contact for the BCC packing.

Proton-conducting superacidic polymer membranes with different fluoroalkyl sulfonate pendants attached to aromatic polymer backbones were synthesized via C-H functionalization and Suzuki coupling reactions. Variation in the chemical structures of the pendant acidic sulfonate moieties and their effects on membrane properties including water uptake, ion exchange capacity, morphology, and proton conductivity were systemically investigated. Membranes containing the short -OCF2SO3H pendant (PSU-S-5) showed a smaller hydrophilic domain size and lower proton conductivity than those containing the longer pendants -OCF2CF2SO3H (PSU-S-1) and -SCF2CF2SO3H (PSU-S-4), likely due to the short chain's less favorable aggregation and lower acidity. Polymer electrolyte membranes with unique branched fluoroalkyl sulfonate pendants (PSU-S-6) gave larger ionic domain sizes, more uniform hydrophilic channels, and higher proton conductivity than samples with analogous linear pendant chains (PSU-S-1), indicating that branched sulfonate structures may be a key future direction in the field of fuel cell membrane.

We report a simple and low-cost strategy to enhance the dielectric permittivity of polystyrene by up to an order of magnitude via incorporating an oligoaniline trimer moiety at the end of the polymer chains. The oligoaniline-capped polystyrene was prepared by a copper-catalyzed click reaction between azide-capped polystyrene and an alkyne-containing aniline trimer, which was doped by different acids. By controlling molecular weight of polystyrene, the end-capped polymers can be induced to form nanoscale oligoaniline-rich domains embedded in an insulating matrix. Under an external electric field, this led to an increase in dielectric polarizability while maintaining a low dielectric loss. At frequencies as high as 0.1 MHz, the dielectric permittivity and dielectric loss (tan δ) were ∼22.8 and ∼0.02, respectively. This strategy may open a new avenue to increasing the dielectric permittivity of many other commodity polymers while maintaining relatively low dielectric loss.

In the photocycle of bacteriorhodopsin at pH 7, a proton is ejected to the extracellular medium during the protonation of Asp-85 upon formation of the M intermediate. The group that releases the ejected proton does not become reprotonated until the prephotolysis state is restored from the N and O intermediates. In contrast, at acidic pH, this proton release group remains protonated to the end of the cycle. Time-resolved Fourier transform infrared measurements obtained at pH 5 and 7 were fitted to obtain spectra of kinetic intermediates, from which the spectra of M and N/O versus unphotolyzed state were calculated. Vibrational features that appear in both M and N/O spectra at pH 7, but not at pH 5, are attributable to deprotonation from the proton release group and resulting structural alterations. Our results agree with the earlier conclusion that this group is a protonated internal water cluster, and provide a stronger experimental basis for this assignment. A decrease in local polarity at the N-C bond of the side chain of Lys-216 resulting from deprotonation of this water cluster may be responsible for the increase in the proton affinity of Asp-85 through M and N/O, which is crucial for maintaining the directionality of proton pumping.

Cbeliji}rniLtr7tvine,In the photocycle of bacteriorhodopsin at neutral pH, the proton transfer fi'om the Schiff base to Asp85 in the L-to-M trarisition causes dcprotonation oF the proton release group (PRG) which is ]ocatcd cTose to the extraceT]ular surface.This leads to unidiTectiona! proton transfer by preventing the reverse proton flows.This prevention is lifted at pH 4-5 below the pKa =--5.7 efthe PRG in M where proton release occurs later after proton uptakc.High proton aMinity to Asp85 at ncutral pH is suppesed to be resulted from structura] rearrangernent induced by the deprotonation of the PRG, in addition to a simpTe elecn'ostatic interaction with the PRG.In the current study, structurul changes were explored in time-resolved FTIR spcctral changes that persisted through the M, N, and O

Na + -NQR is a unique respiratory enzyme that couples the free energy of electron transfer reactions to electrogenic pumping of sodium across the cell membrane. This enzyme is found in many marine and pathogenic bacteria where it plays an analogous role to the H + -pumping complex I. It has generally been assumed that the sodium pump of Na + -NQR operates on the basis of thermodynamic coupling between reduction of a single redox cofactor and the binding of sodium at a nearby site. In this study, we have defined the coupling to sodium translocation of individual steps in the redox reaction of Na + -NQR. Sodium uptake takes place in the reaction step in which an electron moves from the 2Fe-2S center to FMN C , while the translocation of sodium across the membrane dielectric (and probably its release into the external medium) occurs when an electron moves from FMN B to riboflavin. This argues against a single-site coupling model because the redox steps that drive these two parts of the sodium pumping process do not have any redox cofactor in common. The significance of these results for the mechanism of coupling is discussed, and we proposed that Na + -NQR operates through a novel mechanism based on kinetic coupling, mediated by conformational changes.

In the photocycle of bacteriorhodopsin at pH 7, proton release from the proton releasing group (PRG) to the extracellular medium occurs during formation of the M intermediate. This proton release is inhibited at acidic pH, below the pK(a) of the PRG, approximately 6 in M, and instead occurs later in the cycle as the initial state is restored from the O intermediate. Here, structural changes related to deprotonation of the PRG have been investigated by time-resolved FTIR spectroscopy at 25 degrees C. The vibrational features at 2100-1790, 1730-1685, 1661, and 1130-1045 cm(-1) have greater negative intensity in the pure M-minus-BR spectrum and even in the M-minus-BR spectrum, that is present earlier together with the L-minus-BR spectrum, at pH 7, than in the corresponding M-minus-BR spectra at pH 5 or 4. The D212N mutation abolishes the decreases in the intensities of the broad feature between 1730 and 1685 cm(-1) and the band at 1661 cm(-1). The 1730-1685 cm(-1) feature may arise from transition dipole coupling of the backbone carbonyl groups of Glu204, Phe208, Asp212, and Lys216 interacting with Tyr57 and C(15)-H of the chromophore. The 1661 cm(-1) band, which is insensitive to D(2)O substitution, may arise by interaction of the backbone carbonyl of Asp212 with C(15)-H. The 2100-1790 cm(-1) feature with a trough at 1885 cm(-1) could be due to a water cluster. Depletion of these bands upon deprotonation of the PRG is attributable to disruption of a coordinated structure, held in place by interactions of Asp212. Deprotonation of the PRG is also accompanied by disruption of the interaction of the water molecule near Arg82. The liberated Asp212 may stabilize the protonated state of Asp85 and thus confer unidirectionality to the transport.

The Na+-pumping NADH: quinone oxidoreductase (Na+-NQR) is the only respiratory enzyme that operates as a Na+ pump. This redox-driven Na+ pump is amenable to experimental approaches not available for H+ pumps, providing an excellent system for mechanistic studies of ion translocation. An understanding of the internal electron transfer steps and their Na+ dependence is an essential prerequisite for such studies. To this end, we analyzed the reduction kinetics of the wild type Na+-NQR, as well as site-directed mutants of the enzyme, which lack specific cofactors. NADH and ubiquinol were used as reductants in separate experiments, and a full spectrum UV-visible stopped flow kinetic method was employed. The results make it possible to define the complete sequence of redox carriers in the electrons transfer pathway through the enzyme. Electrons flow from NADH to quinone through the FAD in subunit F, the 2Fe-2S center, the FMN in subunit C, the FMN in subunit B, and finally riboflavin. The reduction of the FMNC to its anionic flavosemiquinone state is the first Na+-dependent process, suggesting that reduction of this site is linked to Na+ uptake. During the reduction reaction, two FMNs are transformed to their anionic flavosemiquinone in a single kinetic step. Subsequently, FMNC is converted to the flavohydroquinone, accounting for the single anionic flavosemiquinone radical in the fully reduced enzyme. A model of the electron transfer steps in the catalytic cycle of Na+-NQR is presented to account for the kinetic and spectroscopic data.