Temperature-dependent intermediates in HIV-1 envelope glycoprotein-mediated fusion revealed by inhibitors that target N- and C-terminal helical regions of HIV-1 gp41.

Peptides derived from the N- (N-HR) and C- (C-HR) terminal heptad repeat regions adjacent to the fusion peptide and transmembrane domains, respectively, of human immunodeficiency virus (HIV)-1 gp41 inhibit HIV-1 viral envelope glycoproteins (Env)-mediated cell fusion specifically. The mechanism of HIV-1 Env-mediated cell fusion and its inhibition by agents that target the N- and C-HR regions was investigated. Priming experiments with Env-expressing cells indicate that the N-HR region but not the C-HR region is exposed by treatment with sCD4 at 31 degrees C, whereas both the N- and C-HR regions are exposed at 37 degrees C.

Completely automated, highly error-tolerant macromolecular structure determination from multidimensional nuclear overhauser enhancement spectra and chemical shift assignments.

The major rate-limiting step in high-throughput NMR protein structure determination involves the calculation of a reliable initial fold, the elimination of incorrect nuclear Overhauser enhancement (NOE) assignments, and the resolution of NOE assignment ambiguities. We present a robust approach to automatically calculate structures with a backbone coordinate accuracy of 1.0-1.

Ensemble approach for NMR structure refinement against (1)H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule.

Paramagnetic relaxation enhancement (PRE) measurements on (1)H nuclei have the potential to play an important role in NMR structure determination of macromolecules by providing unique long-range (10-35 A) distance information. Recent methodological advances for covalently attaching paramagnetic groups at specific sites on both proteins and nucleic acids have permitted the application of the PRE to various biological macromolecules. However, because artificially introduced paramagnetic groups are exposed to solvent and linked to the macromolecule by several freely rotatable bonds, they are intrinsically flexible.

Mapping the topology and determination of a low-resolution three-dimensional structure of the calmodulin-melittin complex by chemical cross-linking and high-resolution FTICRMS: direct demonstration of multiple binding modes.

Calmodulin serves as a calcium-dependent regulator in many metabolic pathways and is known to bind with high affinity to various target proteins and peptides. One such target is the small peptide melittin, the principal component of honeybee venom. The calmodulin-melittin system was used as a model system to gain further insight into target recognition of calmodulin.

Are we puppets on a string? The contextual meaning of unconscious expressive cues.

In three studies, the authors show that unconscious expressive cues can lead to opposite evaluations, depending on the context in which they occur. In Study 1, brow (vs. cheek) tension reduced preferences in an easy judgment context but increased preferences in a difficult context.

How much backbone motion in ubiquitin is required to account for dipolar coupling data measured in multiple alignment media as assessed by independent cross-validation?

The magnitude of backbone internal motions in the small protein ubiquitin that needs to be invoked to account for dipolar coupling data measured in multiple alignment media is investigated using an intuitively straightforward approach. This involves simultaneous refinement of the coordinates (against NOE, torsion angle, and dipolar coupling restraints) and optimization of the magnitudes and orientations of the alignment tensors by means of torsion angle simulated annealing and Cartesian space minimization. We show that N-H dipolar couplings in 11 different alignment media and N-C', H(N)-C', and C alpha-C' dipolar coupling in two alignment media can be accounted for, at approximately the level of uncertainty in the experimental data, by a single structure representation.

Why good guys wear white.

Affect is a somewhat abstract concept that is frequently linked to physical metaphor. For example, good is often depicted as light (rather than dark), up (rather than down), and moving forward (rather than backward). The purpose of our studies was to examine whether the association between stimulus brightness and affect is optional or obligatory.

Molecular basis for synergistic transcriptional activation by Oct1 and Sox2 revealed from the solution structure of the 42-kDa Oct1.Sox2.Hoxb1-DNA ternary transcription factor complex.

The Oct and Sox transcription factors control many different aspects of neural development and embryogenesis, often binding to adjacent sites on DNA, and interacting with one another through their DNA binding domains to regulate transcription synergistically. Oct proteins contain two DNA binding domains (POUS and POUHD) connected by a flexible linker, which interact with DNA in a bipartite manner. Residual dipolar coupling measurements on the binary Oct1.

EDTA-derivatized deoxythymidine as a tool for rapid determination of protein binding polarity to DNA by intermolecular paramagnetic relaxation enhancement.

EDTA-derivatized deoxythymidine (dT-EDTA), incorporated into DNA and complexed to Fe2+ in the presence of dithiothreitol, is a widely used reagent for sequence-specific cleavage of duplex DNA. Using HPLC/electrospray mass spectrometry, we show that cleavage is specific to Fe2+, and no cleavage occurs when DNA-EDTA is complexed to other metal ions such as Ca2+, Mn2+, and Fe3+ even after many days. Because dT-EDTA can be incorporated at any desired position of a synthetic oligonucleotide, DNA-EDTA is ideally suited for the measurement of intermolecular paramagnetic relaxation enhancement effects between a paramagnetic ion chelated to DNA-EDTA and a bound protein.

Solution structure of the phosphoryl transfer complex between the signal-transducing protein IIAGlucose and the cytoplasmic domain of the glucose transporter IICBGlucose of the Escherichia coli glucose phosphotransferase system.

The solution structure of the final phosphoryl transfer complex in the glucose-specific arm of the Escherichia coli phosphotransferase system, between enzyme IIAGlucose (IIAGlc) and the cytoplasmic B domain (IIBGlc) of the glucose transporter IICBGlc, has been solved by NMR. The interface (approximately 1200-A2 buried surface) is formed by the interaction of a concave depression on IIAGlc with a convex protrusion on IIBGlc. The phosphoryl donor and acceptor residues, His-90 of IIAGlc and Cys-35 of IIBGlc (residues of IIBGlc are denoted in italics) are in close proximity and buried at the center of the interface.

Covalent trimers of the internal N-terminal trimeric coiled-coil of gp41 and antibodies directed against them are potent inhibitors of HIV envelope-mediated cell fusion.

We have engineered two soluble, covalently linked, trimeric polypeptides, N35CCG-N13 and N34CCG comprising only the internal trimeric coiled-coil of the ectodomain of HIV-1 gp41. Both trimers inhibit human immunodeficiency virus, type 1 (HIV-1) envelope (Env)-mediated cell fusion at nanomolar concentrations by targeting the exposed C-terminal region of the gp41 ectodomain in the prehairpin intermediate state. The IC50 values for N35CCG-N13 and N34CCG are approximately 15 and approximately 95 nM, respectively, in a quantitative vaccinia virus-based reporter gene assay for HIV-1 Env-mediated cell fusion using Env from the T cell tropic strain LAV.

Docking of protein-protein complexes on the basis of highly ambiguous intermolecular distance restraints derived from 1H/15N chemical shift mapping and backbone 15N-1H residual dipolar couplings using conjoined rigid body/torsion angle dynamics.

A simple and reliable method for docking protein-protein complexes using (1)H(N)/(15)N chemical shift mapping and backbone (15)N-(1)H residual dipolar couplings is presented and illustrated with three complexes (EIN-HPr, IIA(Glc)-HPr, and IIA(Mtl)-HPr) of known structure. The (1)H(N)/(15)N chemical shift mapping data are transformed into a set of highly ambiguous, intermolecular distance restraints (comprising between 400 and 3000 individual distances) with translational and some degree of orientational information content, while the dipolar couplings provide information on relative protein-protein orientation. The optimization protocol employs conjoined rigid body/torsion angle dynamics in simulated annealing calculations.

Improving the accuracy of NMR structures of RNA by means of conformational database potentials of mean force as assessed by complete dipolar coupling cross-validation.

The description of the nonbonded contact terms used in simulated annealing refinement can have a major impact on nucleic acid structures generated from NMR data. Using complete dipolar coupling cross-validation, we demonstrate that substantial improvements in coordinate accuracy of NMR structures of RNA can be obtained by making use of two conformational database potentials of mean force: a nucleic acid torsion angle database potential consisting of various multidimensional torsion angle correlations; and an RNA specific base-base positioning potential that provides a simple geometric, statistically based, description of sequential and nonsequential base-base interactions. The former is based on 416 nucleic acid crystal structures solved at a resolution of View Article and Find Full Text PDF

The Xplor-NIH NMR molecular structure determination package.

We announce the availability of the Xplor-NIH software package for NMR biomolecular structure determination. This package consists of the pre-existing XPLOR program, along with many NMR-specific extensions developed at the NIH. In addition to many features which have been developed over the last 20 years, the Xplor-NIH package contains an interface with a new programmatic framework written in C++.

Belief and feeling: evidence for an accessibility model of emotional self-report.

This review organizes a variety of phenomena related to emotional self-report. In doing so, the authors offer an accessibility model that specifies the types of factors that contribute to emotional self-reports under different reporting conditions. One important distinction is between emotion, which is episodic, experiential, and contextual, and beliefs about emotion, which are semantic, conceptual, and decontextualized.

Reweighted atomic densities to represent ensembles of NMR structures.

A reweighted atomic probability density is introduced as a means of representing ensembles of NMR structures in a simple, concise and informative manner. This density is shown to give a better visual representation of molecular structure information than an unweighted density, and should provide a useful interactive graphics tool during the course of iterative NMR structure refinement. The approach is illustrated using several examples.

The epistemic benefits of trait-consistent mood states: an analysis of extraversion and mood.

One must consider both trait and state affect to predict individual differences in emotional processing. The present results document a novel trait-state interaction that is consistent with proposals concerning the epistemic functions of affect (A. R.

Solution structure of the phosphoryl transfer complex between the cytoplasmic A domain of the mannitol transporter IIMannitol and HPr of the Escherichia coli phosphotransferase system.

The solution structure of the complex between the cytoplasmic A domain (IIA(Mtl)) of the mannitol transporter II(Mannitol) and the histidine-containing phosphocarrier protein (HPr) of the Escherichia coli phosphotransferase system has been solved by NMR, including the use of conjoined rigid body/torsion angle dynamics, and residual dipolar couplings, coupled with cross-validation, to permit accurate orientation of the two proteins. A convex surface on HPr, formed by helices 1 and 2, interacts with a complementary concave depression on the surface of IIA(Mtl) formed by helix 3, portions of helices 2 and 4, and beta-strands 2 and 3. The majority of intermolecular contacts are hydrophobic, with a small number of electrostatic interactions at the periphery of the interface.

Molecular basis of sequence-specific single-stranded DNA recognition by KH domains: solution structure of a complex between hnRNP K KH3 and single-stranded DNA.

To elucidate the basis of sequence-specific single-stranded (ss) DNA recognition by K homology (KH) domains, we have solved the solution structure of a complex between the KH3 domain of the transcriptional regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K) and a 10mer ssDNA. We show that hnRNP K KH3 specifically recognizes a tetrad of sequence 5'd-TCCC. The complex is stabilized by a dense network of methyl-oxygen hydrogen bonds involving the methyl groups of three isoleucine residues and the O2 and N3 atoms of the two central cytosine bases.

Episodic and semantic knowledge in emotional self-report: evidence for two judgment processes.

Three studies involving 3 participant samples (Ns = 39, 55, and 53) tested the hypothesis that people retrieve episodic emotion knowledge when reporting on their emotions over short (e.g., last few hours) time frames, but that they retrieve semantic emotion knowledge when reporting on their emotions over long (e.

Theoretical and computational advances in biomolecular NMR spectroscopy.

Recent developments in experimental and computational aspects of NMR spectroscopy have had a significant impact on the accuracy and speed of macromolecular structure determination in solution, particularly with regard to systems of high complexity (such as protein complexes). These include experiments designed to provide long-range orientational and translational restraints, improvements in internal coordinate dynamics used for simulated annealing, and the development of database potentials of mean force to improve the description of the non-bonded contacts.

Chi(1) rotamer populations and angles of mobile surface side chains are accurately predicted by a torsion angle database potential of mean force.

The equilibrium angles and distributions of chi(1) rotamers for mobile surface side chains of the small, 63-residue, B1 domain of protein L have been calculated from the static crystal structure by rigid body/torsion angle simulated annealing using a torsion angle database potential of mean force and compared to those deduced by Monte Carlo analysis of side chain residual dipolar couplings measured in solution. Good agreement between theory and experiment is observed, indicating that for side chains undergoing rotamer averaging that is fast on the chemical shift time scale, the equilibrium angles and distribution of chi(1) rotamers are largely determined by the backbone phi/psi torsion angles.

Attending to the big picture: mood and global versus local processing of visual information.

Two experiments employed image-based tasks to test the hypothesis that happier moods promote a greater focus on the forest and sadder moods a greater focus on the trees. The hypothesis was based on the idea that in task situations, affective cues may be experienced as task-relevant information, which then influences global versus local attention. Using a serial-reproduction paradigm, Experiment 1 showed that individuals in sad moods were less likely than those in happier moods to use an accessible global concept to guide attempts to reproduce a drawing from memory.

Structure and dynamics of KH domains from FBP bound to single-stranded DNA.

Gene regulation can be tightly controlled by recognition of DNA deformations that are induced by stress generated during transcription. The KH domains of the FUSE-binding protein (FBP), a regulator of c-myc expression, bind in vivo and in vitro to the single-stranded far-upstream element (FUSE), 1,500 base pairs upstream from the c-myc promoter. FBP bound to FUSE acts through TFIIH at the promoter.

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41.

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil.