Recombinant expression, purification, and structural analysis of two ectodomains of Syndecan-1.

Syndecan-1 (SDC-1) is an integral membrane heparin sulfate proteoglycan that is involved in inflammatory response, cell-signaling, cell proliferation, and numerous other cell-matrix interactions. Like the other members of the syndecan family, very little is known about structural conformations and dynamics of SDC-1. A majority of interactions occur through the extracellular ectodomain, therefore we have dedicated our research efforts to the study this specific portion of SDC-1. The ectodomain is often shed from the cell surface due to various stimuli. The released fragment has already been used as a useful biomarker for prognosis of some diseases and cancers. SDC-1 can be cleaved in different locations depending on the sheddase, generating soluble shed ectodomains that can be carried away in blood sera. In this study, we focus specifically on two main cleavage fragments that can be generated. We show the first successful expression and purification of recombinant SDC-1 ectodomains. Production of SDC-1 in E. coli allows the production of the core protein without risking heterogeneous post-translational modifications such as glycosylation, allowing a certain level of control over protein homogeneity that is not possible in mammalian expression. An expression vector was used to generate two different fusion proteins consisting of a His-tag and a TEV cleavage site for the removal of the fusion partner. SDS-PAGE was used to track the expression as well as the purification. Masses of the isolated proteins were determined using mass spectrometry and the purity and homogeneity were evaluated by solution NMR.

Expression, purification, and structural analysis of the full-length human integral membrane protein γ-sarcoglycan.

Mutation of the gene encoding γ-sarcoglycan (SGCG), an integral membrane protein responsible for maintaining the integrity of the muscle cell sarcolemma, results in Limb-Girdle Muscular Dystrophy (LGMD), a congenital disease with no current treatment options. This member of the sarcoglycan glycoprotein family is a vital component of the Dystrophin Complex, which together facilitate normal muscle function. However, very little is known about the structure and dynamics of these proteins, and of membrane glycoproteins in general. This is due to a number of factors, including their complexity, heterogeneity and highly-specific native environments. The expression, purification, and structural study of membrane proteins is further impeded by their hydrophobic nature and consequent propensity to aggregate in aqueous solutions. Here, we report the first successful expression and purification of milligram quantities of full-length recombinant SGCG, utilizing fusion protein-guided overexpression to inclusion bodies in Escherichia coli. Purification of SGCG from the fusion protein, TrpΔLE, was facilitated using chemical cleavage. Cleavage products were then isolated by size-exclusion chromatography. Successful purification of the protein was confirmed using SDS-PAGE and mass spectroscopy. Finally, solution nuclear magnetic resonance spectroscopy of uniformly 15N-labeled SGCG in detergent environments was performed, yielding the first spectra of the full-length membrane glycoprotein, SGCG. These results represent the initial structural studies of SGCG, laying the foundation for further investigation on the interaction and dynamics of other integral membrane proteins. More specifically, this data allows for opportunities in the future for enhanced treatment modalities and cures for LGMD.

Risk assessment communication difficulties: An empirical examination of the effects of categorical versus probabilistic risk communication in sexually violent predator decisions.

Expert testimony concerning risk and its communication to the trier of fact has important implications for some of the most significant legal decisions. In a simulated sexual violent predator hearing, we examined how mock jurors interpret and use recidivism risk expert testimony communicated either categorically, using verbal labels, or probabilistically, using numeric values. Based upon the STATIC-99R, we compared mock jurors' decision-making and verdicts when we manipulated the style of risk communication across four different risk levels. In terms of verdict decisions, we found that higher risk levels were associated with more commitment decisions, but that this relationship only existed for the categorical risk-communication format. We also replicated previous research demonstrating that participants overestimate recidivism risk in general, especially when higher risk is communicated categorically. Finally, our participants did not differentiate well between the four levels of risk offered, instead apparently employing a more simplistic dichotomy between "low" or "high" risk for both their verdict decisions and their thresholds for commitment. The legal and policy implications of our findings are discussed, as well as suggestions for more effective presentation of expert risk testimony.

Investigating the subjective reports of rejection processes in the word frequency mirror effect.

We sought to systematically investigate how participants subjectively classify the basis of their recognition memory judgments for low and high word frequency items. We found that participants more often reported rejection processes related to the increased perceived memorability for unstudied low word frequency items (relative to high word frequency items), rather than classifying their decision on a lack of familiarity. Experiment 2 replicated this pattern and demonstrated context variability and word frequency independently influenced the subjective classifications for correct rejections. Results of Experiment 3 revealed that these differences are dependent upon having experience with both low and high frequency items. Overall, these data suggest participants' rejection of low frequency items is more strongly related to judgments of perceived memorability, but only when they are presented in the context of high frequency items. The results are discussed in relation to distinctiveness and expected memorability.

Three-dimensional structure and interaction studies of hepatitis C virus p7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine by solution nuclear magnetic resonance.

Hepatitis C virus (HCV) protein p7 plays an important role in the assembly and release of mature virus particles. This small 63-residue membrane protein has been shown to induce channel activity, which may contribute to its functions. p7 is highly conserved throughout the entire range of HCV genotypes, which contributes to making p7 a potential target for antiviral drugs. The secondary structure of p7 from the J4 genotype and the tilt angles of the helices within bilayers have been previously characterized by nuclear magnetic resonance (NMR). Here we describe the three-dimensional structure of p7 in short chain phospholipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine) micelles, which provide a reasonably effective membrane-mimicking environment that is compatible with solution NMR experiments. Using a combination of chemical shifts, residual dipolar couplings, and PREs, we determined the structure of p7 using an implicit membrane potential combining both CS-Rosetta decoys and Xplor-NIH refinement. The final set of structures has a backbone root-mean-square deviation of 2.18 Å. Molecular dynamics simulations in NAMD indicate that several side chain interactions might be taking place and that these could affect the dynamics of the protein. In addition to probing the dynamics of p7, we evaluated several drug-protein and protein-protein interactions. Established channel-blocking compounds such as amantadine, hexamethylene amiloride, and long alkyl chain iminosugar derivatives inhibit the ion channel activity of p7. It has also been shown that the protein interacts with HCV nonstructural protein 2 at the endoplasmic reticulum and that this interaction may be important for the infectivity of the virus. Changes in the chemical shift frequencies of solution NMR spectra identify the residues taking part in these interactions.

In Vitro Glycosylation of the Membrane Protein γ-Sarcoglycan in Nanodiscs.

Membrane glycoproteins are proteins that reside in the membranes of cells and are post-translationally modified to have sugars attached to their amino acid side chains. Studies of this subset of proteins in their native states are becoming more important since they have been linked to numerous human diseases. However, these proteins are difficult to study due to their hydrophobic nature and their propensity to aggregate. Using membrane mimetics allows us to solubilize these proteins, which, in turn, allows us to perform glycosylation in vitro to study the effects of the modification on protein structure, dynamics, and interactions. Here, the membrane glycoprotein γ-sarcoglycan was incorporated into nanodiscs composed of long-chain lipids and membrane scaffold proteins to perform N-linked glycosylation in which an enzyme attaches a sugar to the asparagine side chain within the glycosylation site. We previously performed glycosylation of membrane proteins in vitro when the protein had been solubilized using different detergents and short-chain lipids. This work demonstrates successful glycosylation of a full-length membrane protein in nanodiscs providing a more biologically relevant sample to study the effects of the modification.

Secondary structure, dynamics, and architecture of the p7 membrane protein from hepatitis C virus by NMR spectroscopy.

P7 is a small membrane protein that is essential for the infectivity of hepatitis C virus. Solution-state NMR experiments on p7 in DHPC micelles, including hydrogen/deuterium exchange, paramagnetic relaxation enhancement and bicelle 'q-titration,' demonstrate that the protein has a range of dynamic properties and distinct structural segments. These data along with residual dipolar couplings yield a secondary structure model of p7. We were able to confirm previous proposals that the protein has two transmembrane segments with a short interhelical loop containing the two basic residues K33 and R35. The 63-amino acid protein has a remarkably complex structure made up of seven identifiable sections, four of which are helical segments with different tilt angles and dynamics. A solid-state NMR two-dimensional separated local field spectrum of p7 aligned in phospholipid bilayers provided the tilt angles of two of these segments. A preliminary structural model of p7 derived from these NMR data is presented.

Comparative NMR studies demonstrate profound differences between two viroporins: p7 of HCV and Vpu of HIV-1.

The p7 protein from hepatitis C virus and the Vpu protein from HIV-1 are members of the viroporin family of small viral membrane proteins. It is essential to determine their structures in order to obtain an understanding of their molecular mechanisms and to develop new classes of anti-viral drugs. Because they are membrane proteins, it is challenging to study them in their native phospholipid bilayer environments by most experimental methods. Here we describe applications of NMR spectroscopy to both p7 and Vpu. Isotopically labeled p7 and Vpu samples were prepared by heterologous expression in bacteria, initial isolation as fusion proteins, and final purification by chromatography. The purified proteins were studied in the model membrane environments of micelles by solution NMR spectroscopy and in aligned phospholipid bilayers by solid-state NMR spectroscopy. The resulting structural findings enable comparisons to be made between the two proteins, demonstrating that they have quite different architectures. Most notably, Vpu has one trans-membrane helix and p7 has two trans-membrane helices; in addition, there are significant differences in the structures and dynamics of their internal loop and terminal regions.

EF-hand protein, EfhP, specifically binds Ca2+ and mediates Ca2+ regulation of virulence in a human pathogen Pseudomonas aeruginosa.

Calcium (Ca2+) is well known as a second messenger in eukaryotes, where Ca2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca2+ signaling, little is known about the mechanisms of bacterial Ca2+ signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+ with 13.7 µM affinity, and that mutations at the +X and -Z positions within each or both EF-hand motifs abolished Ca2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca2+-dependent manner, however no such response was detected in the mutated proteins. 15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca2+ binding abolished Ca2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca2+ sensor.

Nanodiscs versus macrodiscs for NMR of membrane proteins.

It is challenging to find membrane mimics that stabilize the native structures, dynamics, and functions of membrane proteins. In a recent advance, nanodiscs have been shown to provide a bilayer environment compatible with solution NMR. We show that increasing the lipid to "belt" peptide ratio expands their diameter, slows their reorientation rate, and allows the protein-containing discs to be aligned in a magnetic field for oriented sample solid-state NMR. The spectroscopic properties of membrane proteins with one to seven transmembrane helices in q = 0.1 isotropic bicelles, ~10 nm diameter isotropic nanodiscs, ~30 nm diameter magnetically aligned macrodiscs, and q = 5 magnetically aligned bicelles are compared.

In Vitro Glycosylation of Membrane Proteins Using N-Glycosyltransferase.

Glycoproteins are post-translationally modified proteins that take part in nearly every biological process and make up a large percent of the proteome. N-Linked glycosylation can be performed by N-glycosyltransferase (NGT), which recognizes the consensus amino acid sequence, -Asn-X-Ser/Thr- (NXT), within the protein. The enzyme catalyzes glycosidic bond formation between the oligosaccharide donor, containing nucleoside phosphatase, and the amide nitrogen of the asparagine residue. The attachment of the sugar moiety can influence physiological and biological properties of the protein by affecting their folding, modulating interactions with other biomolecules, and modifying their functions at the cellular level. We are specifically interested in the properties of membrane glycoproteins, which are key components in a number of different disease states. Therefore, the use of in vitro protein glycosylation can help further evaluate the effects of the properties for these important macromolecules. In vitro studies of N-linked glycosylation were done in a stepwise fashion in a membrane-mimetic environment to confirm that the methods for glycosylating soluble proteins could be applicable to membrane proteins. Detergent and lipid systems were used since hydrophobic peptides and membrane proteins are insoluble in aqueous solvents. The stepwise method consisted of the glycosylation of a soluble 7-residue peptide, a hydrophobic WALP-NVT peptide, and a γ-sarcoglycan membrane protein, all of which contained the glycosylation site Asn-Val-Thr (NVT). Glycosylation of the samples was performed using Escherichia coli-expressed NGT from the Actinobacillus pleuropneumoniae genome, and a single sugar moiety of glucose, provided from a nucleotide-linked donor, was added to the glycosylation site. Gel electrophoresis, mass spectrometry, and NMR studies were used for the detection of glycosyltransferase activity and to show the attachment of a single glucose molecule. Our experiments demonstrated that small or large membrane proteins that contain an N-glycosylation consensus sequence can be glycosylated by NGT in membrane-mimetic environments.

Structural characterization of two pore-forming peptides: consequences of introducing a C-terminal tryptophan.

Synthetic channel-forming peptides that can restore chloride conductance across epithelial membranes could provide a novel treatment of channelopathies such as cystic fibrosis. Among a series of 22-residue peptides derived from the second transmembrane segment of the glycine receptor alpha(1)-subunit (M2GlyR), p22-S22W (KKKKP ARVGL GITTV LTMTT QW) is particularly promising with robust membrane insertion and assembly. The concentration to reach one-half maximal short circuit current is reduced to 45 +/- 6 microM from that of 210 +/- 70 microM of peptide p22 (KKKKP ARVGL GITTV LTMTT QS). However, this is accompanied with nearly 50% reduction in conductance. Toward obtaining a molecular level understanding of the channel activities, we combine information from solution NMR, existing biophysical data, and molecular modeling to construct atomistic models of the putative pentameric channels of p22 and p22-S22W. Simulations in membrane bilayers demonstrate that these structural models, even though highly flexible, are stable and remain adequately open for ion conductance. The membrane-anchoring tryptophan residues not only rigidify the whole channel, suggesting increased stability, but also lead to global changes in the pore profile. Specifically, the p22-S22W pore has a smaller opening on average, consistent with lower measured conductance. Direct observation of several incidences of chloride transport suggests several qualitative features of how these channels might selectively conduct anions. The current study thus helps to rationalize the functional consequences of introducing a single C-terminal tryptophan. Availability of these structural models also paves the way for future work to rationally modify and improve M2GlyR-derived peptides toward potential peptide-based channel replacement therapy.

NMR studies of p7 protein from hepatitis C virus.

The p7 protein of hepatitis C virus (HCV) plays an important role in the viral lifecycle. Like other members of the viroporin family of small membrane proteins, the amino acid sequence of p7 is largely conserved over the entire range of genotypes, and it forms ion channels that can be blocked by a number of established channel-blocking compounds. Its characteristics as a membrane protein make it difficult to study by most structural techniques, since it requires the presence of lipids to fold and function properly. Purified p7 can be incorporated into phospholipid bilayers and micelles. Initial solid-state nuclear magnetic resonance (NMR) studies of p7 in 14-O-PC/6-O-PC bicelles indicate that the protein contains helical segments that are tilted approximately 10 degrees and 25 degrees relative to the bilayer normal. A truncated construct corresponding to the second transmembrane domain of p7 is shown to have properties similar to those of the full-length protein, and was used to determine that the helix segment tilted at 10 degrees is in the C-terminal portion of the protein. The addition of the channel blocker amantadine to the full-length protein resulted in selective chemical shift changes, demonstrating that NMR has a potential role in the development of drugs targeted to p7.

Threat-related processing supports prospective memory retrieval for people with obsessive tendencies.

Obsessive-compulsive disorder can result in a variety of deficits to cognitive performance, including negative consequences for attention and memory performance. The question addressed in the current study concerned whether this disorder influenced performance in an event-based prospective memory task. The results from a subclinical population indicated that, relative to non-anxious controls and mildly depressed controls, people with obsessive-compulsive tendencies (washing compulsions) incur decrements in remembering to respond to cues related to a neutral intention (respond to animals). This deficit was ameliorated by giving the subclinical group an intention about a threat-related category (respond to bodily fluids) and cueing them with concepts that they had previously rated as particularly disturbing to them. Thus, their normal attentional bias for extended processing of threat-related information overcame their natural deficit in event-based prospective memory.

The valence of event-based prospective memory cues or the context in which they occur affects their detection.

Event-based prospective memory tasks entail detecting cues or reminders in our environment related to previously established intentions. If they are detected at an opportune time, then the intention can be fulfilled. In Experiments 1a-1c, we gave people 3 different nonfocal intentions (e.g., respond to words denoting animals) and discovered that negatively valenced cues delivered the intention to mind less frequently than positively valenced cues. In Experiment 2, this effect was extended to valenced and neutral sentential contexts with convergent results that cues embedded in negatively valenced sentences evoked remembering the intention less often than in positive contexts. In addition, both classes of valence caused the intention to be forgotten more often than a more neutral context. We propose that valence has the ability to usurp attentional resources that otherwise would have supported successful prospective memory performance.

Learning is impaired by activated intentions.

Two experiments examined the task interference that sometimes accrues from having an intention. In standard prospective memory tasks, latency is often slower to an ongoing task performed concurrently with having an intention than it is when no intention is given. If the locus of this slowing resulted from different attentional allocation policies in the two cases, we predicted that the process of learning a word list would be impaired if participants had an intention rather than if they did not. Four different event-based prospective memory tasks were used in Experiment 1 to demonstrate that worse free recall of a word list resulted when studied with a concurrent intention than with a control condition that had no intention. In that experiment, linking an intention to a distal context that was to occur after learning did not impair free recall. Two time-based tasks were used in Experiment 2 to demonstrate that possessing a time-based prospective memory also hinders learning, unless the intention is linked to a future context that is expected to occur after the study session. In the latter case, no impairment was obtained.

Comparing older and younger adults in an event-based prospective memory paradigm containing an output monitoring component.

Two experiments with younger and older adults were conducted to investigate the output-monitoring component of event-based prospective memory. In the standard form of the task, participants must remember to press a key when a certain class of items is encountered. To evaluate output monitoring, event-based cues were repeated and participants were asked to press a different key if they could remember that an earlier response was made to a particular cue. Younger adults forgot fewer of their successful responses, but displayed a distinct bias to claim that they had responded earlier when actually they had forgotten to respond. By contrast, older adults displayed this bias much less frequently. Elaborated responding to cues had the effect of improving the performance of younger, but not older adults. The results are discussed in terms of natural repetitions and omission errors that might be made in everyday prospective memory tasks.

Source memory in the absence of successful cued recall.

Five experiments were conducted to address the question of whether source information could be accessed in the absence of being able to recall an item. The authors used a paired-associate learning paradigm in which cue-target word pairs were studied, and target recall was requested in the presence of the cue. When target recall failed, participants were asked to make a source judgment of whether a man or woman spoke the unrecalled item. In 3 of the 5 experiments, source accuracy was at or very close to chance. By contrast, if cue-target pairs were studied multiple times or participants knew in advance of learning that a predictive judgment would be required, then predictive source accuracy was well above chance. These data are suggestive that context information may not play a very large role in metacognitive judgments such as feeling-of-knowing ratings or putting one into a tip-of-the-tongue state without strong and specific encoding procedures. These same results also highlight the important role that item memory plays in retrieving information about the context in which an item was experienced.

On rejecting emotional lures created by phonological neighborhood activation.

The authors conducted 2 experiments to assess how phonologically related lures are rejected in a false memory paradigm. Some phonological lures were emotional (i.e., taboo) words, and others were not. The authors manipulated the presence of taboo items on the study list and reduced the ability to use controlled rejection strategies by dividing attention and forcing a short response deadline. The results converge on the idea that participants reduce false alarms to emotional lures by setting more stringent recognition criteria for these items based on their expected memorability. Additionally, emotional lures are less familiar than nonemotional lures because emotional lures have affective and semantic features that mismatch studied nonemotional items.

Concreteness and item-to-list context associations in the free recall of items differing in context variability.

Context variability can be defined as the number of preexperimental contexts in which a given concept appears. Following M. Steyvers and K. J. Malmberg's (2003) work, the authors have shown that concepts that are experienced in fewer preexperimental contexts generally are better remembered in episodic memory tasks than concepts that are experienced in a greater number of preexperimental contexts. The purpose of this article is to demonstrate that low context variability confers its memorial advantage because of stronger item-to-list context associations as compared with high context variability. Three experiments that use environmental context changes from study to test demonstrate that the low context variability advantage is eliminated when item-to-list context associations are not available because of environmental changes at test. In addition, the low context variability advantage is eliminated when inward processing at study prevents the formation of item-to-list context associations.