Conformational Distribution of a Multidomain Protein Measured by Single-Pair Small-Angle X-ray Scattering.

The difficulty in evaluating the conformational distribution of proteins in solution often hinders mechanistic insights. One possible strategy for visualizing conformational distribution is distance distribution measurement by single-pair small-angle X-ray scattering (SAXS), in which the scattering interference from only a specific pair of atoms in the target molecule is extracted. Despite this promising concept, with few applications in synthetic small molecules and DNA, technical difficulties have prevented its application in protein conformational studies. This study used a synthetic tag to fix the lanthanide ion at desired sites on the protein and used single-pair SAXS with contrast matching to evaluate the conformational distribution of the multidomain protein enzyme MurD. These data highlighted the broad conformational and ligand-driven distribution shifts of MurD in solution. This study proposes an important strategy in solution structural biology that targets dynamic proteins, including multidomain and intrinsically disordered proteins.

Conformational ensemble of a multidomain protein explored by Gd3+ electron paramagnetic resonance.

Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.

The role of peer reward and punishment for public goods problems in a localized society.

Cooperation in social dilemmas can be sustained if individuals are effectively rewarded or punished from peers within the group. However, as group size increases, we inevitably face localization, in which a global group is divided into several localized groups. In such societies, members can reward and punish only neighbors within the same localized group, while cooperation for social dilemmas should be solved through global group involvement. In this situation, the global group and the local group are not always equal in terms of welfare, and situations can arise in which cooperation is beneficial for the global group but not for the local group. We predict that in such a locally inefficient situation, peer reward and punishment cannot function to sustain global cooperation. We conducted an experiment in which 16 group members played a public goods game incorporating peer reward and punishment. We manipulated the range of peer reward and punishment (only local members/all global members) and payoff structure (locally efficient/locally inefficient). We found that high cooperation was not achieved and that peer reward and punishment did not function when, and only when, the group was divided into localized groups and the payoff structure was locally inefficient.

Is human life worth peanuts? Risk attitude changes in accordance with varying stakes.

Risk aversion is well-known as a general and robust characteristic of people's decision making: people are less likely to gamble when they are unsure if they will obtain the expected value of the bet made. The "peanuts effect" is, however, an exception to this general rule. The "peanuts effect," which states that people are more willing to gamble when playing for "peanuts" (a small outcome), has been stably observed in the context of a small monetary stake. We conducted two types of experiments to verify whether the peanuts effect still occurred when the type of stakes changed. We had two main findings. On the one hand, people tended to gamble more for a qualitatively smaller value when the stake was material in nature, meaning that the "peanuts effect" occurred with a qualitatively low stake. On the other hand, people were willing to take a risk for a qualitatively larger value when the stake was a human life: this is the opposite phenomenon of the "peanuts effect."

Incidence of venous thromboembolism in psychiatric inpatients: a chart review.

PURPOSE: Venous thromboembolism (VTE) is the combination of pulmonary embolism (PE) and deep vein thrombosis. In recent years, VTE has been gaining attention in the field of psychiatry as it can cause sudden deaths in patients hospitalized in psychiatric departments. The purpose of this study was to investigate the incidence of VTE in psychiatric inpatients using contrast-enhanced computed tomography (CT). PATIENTS AND METHODS: At the psychiatric department of the Akita University Hospital, NANOPIA® D-dimer was measured in patients with suspected symptomatic VTE or believed to be at risk for asymptomatic VTE. A follow-up contrast-enhanced CT was also performed in cases of D-dimer values over 1 µg/mL. Patients diagnosed with VTE based on contrast-enhanced CT during hospitalizations between May 1, 2009 and April 30, 2017 were analyzed. VTE incidence was compared in restrained and unrestrained catatonic and noncatatonic patients. We also investigated whether VTE was symptomatic or asymptomatic as well as its outcomes. RESULTS: The overall incidence of VTE was 2.3% (39/1,681) in the 8-year period. VTE was observed in 61.1% (11/18) of catatonic patients, 4.1% (11/270) of noncatatonic restrained patients, and 1.2% (17/1,393) of noncatatonic unrestrained patients. PE was observed in 76.9% (30/39) of VTE patients and 97.4% (38/39) of VTE patients were asymptomatic. Recovery was achieved in all cases of VTE treated with anticoagulation therapy. CONCLUSION: These results indicate that the risk of VTE is high in psychiatric inpatients and that PE is common in these population. The data may also suggest that contrast-enhanced CT is important in surveying thrombus in suspected cases of VTE. In the psychiatric field, proper attention must be given to VTE, regardless of the presence or absence of catatonia or restraint, particularly given that PE was observed in more than 75% of cases of VTE.

Punishing second-order free riders before first-order free riders: The effect of pool punishment priority on cooperation.

Second-order free riders, who do not owe punishment cost to first-order free riders in public goods games, lead to low cooperation. Previous studies suggest that for stable cooperation, it is critical to have a pool punishment system with second-order punishment, which gathers resources from group members and punishes second-order free riders as well as first-order free riders. In this study, we focus on the priority of punishment. We hypothesize that the pool punishment system that prioritizes second-order punishment is more likely to achieve cooperation than the system that prioritizes first-order punishment, because the former is more likely to obtain sufficient punishment resources. In the experiments, we compare four pool punishment systems: 1To2 (first-order punishment to second-order punishment), 2To1 (second-order punishment to first-order punishment), 1ONLY (first-order punishment only), and 2ONLY (second-order punishment only). We find that the 2To1 and 2ONLY systems can receive more support than the 1To2 and 1ONLY systems and only the 2To1 system can achieve high cooperation. However, the effect of priority of second-order punishment is observed only when the punishment ratio (PR) is low (Experiment 1), not high (Experiment 2), in which the punishment resource is relatively abundant.

Solving the second-order free rider problem in a public goods game: An experiment using a leader support system.

Punishment of non-cooperators-free riders-can lead to high cooperation in public goods games (PGG). However, second-order free riders, who do not pay punishment costs, reduce the effectiveness of punishment. Here we introduce a "leader support system," in which one group leader can freely punish group followers using capital pooled through the support of group followers. In our experiment, participants engage in three stages repeatedly: a PGG stage in which followers decide to cooperate for their group; a support stage in which followers decide whether to support the leader; and a punishment stage in which the leader can punish any follower. We compare a support-present condition with a no-support condition, in which there is an external source for the leader's punishment. The results show that punishment occurs more frequently in the support-present condition than the no-support condition. Within the former, both higher cooperation and higher support for a leader are achieved under linkage-type leaders-who punish both non-cooperators and non-supporters. In addition, linkage-type leaders themselves earn higher profits than other leader types because they withdraw more support. This means that leaders who effectively punish followers could increase their own benefits and the second-order free rider problem would be solved.

Institutionalize Reciprocity to Overcome the Public Goods Provision Problem.

Cooperation is fundamental to human societies, and one of the important paths for its emergence and maintenance is reciprocity. In prisoner's dilemma (PD) experiments, reciprocal strategies are often effective at attaining and maintaining high cooperation. In many public goods (PG) games or n-person PD experiments, however, reciprocal strategies are not successful at engendering cooperation. In the present paper, we attribute this difficulty to a coordination problem against free riding among reciprocators: Because it is difficult for the reciprocators to coordinate their behaviors against free riders, this may lead to inequality among players, which will demotivate them from cooperating in future rounds. We propose a new mechanism, institutionalized reciprocity (IR), which refers to embedding the reciprocal strategy as an institution (i.e., institutionalizing the reciprocal strategy). We experimentally demonstrate that IR can prevent groups of reciprocators from falling into coordination failure and achieve high cooperation in PG games. In conclusion, we argue that a natural extension of the present study will be to investigate the possibility of IR to serve as a collective punishment system.

Ligand-driven conformational changes of MurD visualized by paramagnetic NMR.

Proteins, especially multi-domain proteins, often undergo drastic conformational changes upon binding to ligands or by post-translational modifications, which is a key step to regulate their function. However, the detailed mechanisms of such dynamic regulation of the functional processes are poorly understood because of the lack of an efficient tool. We here demonstrate detailed characterization of conformational changes of MurD, a 47 kDa protein enzyme consisting of three domains, by the use of solution NMR equipped with paramagnetic lanthanide probe. Quantitative analysis of pseudocontact shifts has identified a novel conformational state of MurD, named semi-closed conformation, which is found to be the key to understand how MurD regulates the binding of the ligands. The modulation of the affinity coupled with conformational changes accentuates the importance of conformational state to be evaluated in drug design.

Residual effects of zolpidem, triazolam, rilmazafone and placebo in healthy elderly subjects: a randomized double-blind study.

With current hypnotic agents, next-day residual effects are a common problem. The purpose of the present study was to evaluate the residual effects of the commercially available hypnotics - zolpidem, triazolam, and rilmazafone - on the physical and cognitive functions of healthy elderly people in the early morning and the day following drug administration. In this study, the next-day residual effects of zolpidem, triazolam, and rilmazafone, following bedtime dosing in elderly subjects, were evaluated. Women (n = 11) and men (n = 2) aged 60-70 years received a single dose (at 23:00) of one of these, zolpidem 5 mg, triazolam 0.125 mg, rilmazafone 1 mg and placebo in a randomized, double-blind, crossover design. Measures of objective parameters and psychomotor performances (Timed up and Go test, Functional Reach Test, body sway test, critical flicker fusion test, simple discrimination reaction test, short-term memory test) and subjective ratings were obtained at 04:00, 07:00, and the next time of the day. All hypnotics were generally well tolerated; there were no serious adverse side effects and no subjects discontinued the evaluations. Compared to placebo, zolpidem and rilmazafone had good results on the Functional Reach Test. Although subjective assessments tended to be poor in the early morning, rilmazafone significantly improved the body sway test in the other hypnotics. A single dose of zolpidem 5 mg and triazolam 0.125 mg did not have any next-day residual effects on healthy elderly subjects. Residual effects appeared to be related to the compound's half-life and the dose used. Rilmazafone 1 mg exhibited steadiness in static and dynamic balance and seemed to be more favorable for the elderly with early morning awakening.

Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications.

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-ß-D-glucoside (ß-OG), n-dodecyl-ß-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.

Synthesis of uniformly deuterated n-dodecyl-ß-D-maltoside (d39-DDM) for solubilization of membrane proteins in TROSY NMR experiments.

This work reports the first synthesis of uniformly deuterated n-dodecyl-ß-D-maltoside (d39-DDM). DDM is a mild non-ionic detergent often used in the extraction and purification of membrane proteins and for solubilizing them in experimental studies of their structure, dynamics and binding of ligands. We required d39-DDM for solubilizing large α-helical membrane proteins in samples for [(15)N-(1)H]TROSY (transverse relaxation-optimized spectroscopy) NMR experiments to achieve the highest sensitivity and best resolved spectra possible. Our synthesis of d39-DDM used d7-D-glucose and d25-n-dodecanol to introduce deuterium labelling into both the maltoside and dodecyl moieties, respectively. Two glucose molecules, one converted to a glycosyl acceptor with a free C4 hydroxyl group and one converted to a glycosyl donor substituted at C1 with a bromine in the α-configuration, were coupled together with an α(1 → 4) glycosidic bond to give maltose, which was then coupled with n-dodecanol by its substitution of a C1 bromine in the α-configuration to give DDM. (1)H NMR spectra were used to confirm a high level of deuteration in the synthesized d39-DDM and to demonstrate its use in eliminating interfering signals from TROSY NMR spectra of a 52-kDa sugar transport protein solubilized in DDM.

How can group experience influence the cue priority? A re-examination of the ambiguity-ambivalence hypothesis.

Since the discovery of the "framing effect" by Kahneman and Tversky, the sensitivity of the "framing effect" - its appearance and in some cases its disappearance - has long been an object of study. However there is little agreement as to the reasons for this sensitivity. The "ambiguity-ambivalence hypothesis" (Wang, 2008) aims to systematically explain the sensitivity of this effect by paying particular attention to people's cue priority: it states that the framing effect occurs when verbal framing is used to compensate for the absence of higher prioritized decision cues. The main purpose of our study is to examine and develop this hypothesis by examining cue priority given differences in people's "group experience." The main result is that the framing effect is absent when the choice problem is presented in a group context that reflects the actual size of the group that the participant has had experience with. Thus, in order to understand the choices that people make in life and death decisions, it is important to incorporate the decision maker's group experience explicitly into the ambiguity-ambivalence hypothesis.

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe.

A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligand-protein complex is determined. PRE is an isotropic paramagnetic effect observed within 30 Å from the lanthanide ion, and is utilized for the ligand screening in the present study. PCS is an anisotropic paramagnetic effect providing long-range (~40 Å) distance and angular information on the observed nuclei relative to the paramagnetic lanthanide ion, and utilized for the structure determination of the ligand-protein complex. Since a two-point anchored lanthanide-binding peptide tag is utilized for fixing the lanthanide ion to the target protein, this screening method can be generally applied to non-metal-binding proteins. The usefulness of this strategy was demonstrated in the case of the growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain and its low- and high-affinity ligands.

Chemical synthesis, folding, and structural insights into O-fucosylated epidermal growth factor-like repeat 12 of mouse Notch-1 receptor.

Notch receptors are cell surface glycoproteins that play key roles in a number of developmental cascades in metazoa. The extracellular domains of Notch-1 receptors are composed of 36 tandem epidermal growth factor (EGF)-like repeats, many of which are modified at highly conserved consensus sites by an unusual form of O-glycan, with O-fucose. The O-fucose residues on certain EGF repeats may be elongated. In mammalian cells this can be a tetrasaccharide, Siaα2,3Galß1,4GlcNAcß1,3Fucα1→. This elongation process is initiated by the action of O-fucose-specific ß1,3 N-acetylglucosaminyltransferases of the Fringe family. There is evidence that the addition of GlcNAc by Fringe serves as an essential modulator of the interaction of Notch with its ligands and the triggering of activation. Here we describe the efficient synthesis, folding, and structural characterization of EGF repeat 12 (EGF 12) of a mouse Notch-1 receptor bearing different O-fucose glycan chains. We demonstrate that the three disulfide bonds, Cys(456)-Cys(467) (C1-C3), Cys(461)-Cys(476) (C2-C4), and Cys(478)-Cys(487) (C5-C6) were correctly formed in the nonglycosylated as well as the O-fucosylated forms of EGF 12. Three-dimensional structural studies by NMR reveal that the methyl group of fucose is in close contact with ILe(475), Met(477), Pro(478) residues and this stabilizes the conformation of the antiparallel ß-sheet of EGF 12. The addition of the GlcNAc residue on O-fucosylated EGF 12 induces a significant conformational change in the adjacent tripeptide sequence, Gln(462)Asn(463)Asp(464), which is a motif involved in the natural, enzymatic O-fucosylation at the conserved site (Cys(461)X(4)Ser/ThrCys(467)).