Comparison of terbium (III) luminescence enhancement in mutants of EF hand calcium binding proteins.

The luminescent isomorphous Ca2+ analogue, Tb3+, can be bound in the 12-amino acid metal binding sites of proteins of the EF hand family, and its luminescence can be enhanced by energy transfer from a nearby aromatic amino acid. Tb3+ can be used as a sensitive luminescent probe of the structure and function of these proteins. The effect of changing the molecular environment around Tb3+ on its luminescence was studied using native Cod III parvalbumin and site-directed mutants of both oncomodulin and calmodulin. Titrations of these proteins showed stoichiometries of fill corresponding to the number of Ca2+ binding loops present. Tryptophan in binding loop position 7 best enhanced Tb3+ luminescence in the oncomodulin mutant Y57W, as well as VU-9 (F99W) and VU-32 (T26W) calmodulin. Excitation spectra of Y57F, F102W, Y65W oncomodulin, and Cod III parvalbumin revealed that the principal Tb3+ luminescence donor residues were phenylalanine or tyrosine located in position 7 of a loop, despite the presence of other nearby donors, including tryptophan. Spectra also revealed conformational differences between the Ca2+- and Tb(3+)-bound forms. An alternate binding loop, based on Tb3+ binding to model peptides, was inserted into the CD loop of oncomodulin by cassette mutagenesis. The order of fill of Tb3+ in this protein reversed, with the mutated loop binding Tb3+ first. This indicates a much higher affinity for the consensus-based mutant loop. The mutant loop inserted into oncomodulin had 32 times more Tb3+ luminescence than the identical synthetic peptide, despite having the same donor tryptophan and metal binding ligands. In this paper, a ranking of sensitivity of luminescence of bound Tb3+ is made among this subset of calcium binding proteins. This ranking is interpreted in light of the structural differences affecting Tb3+ luminescence enhancement intensity. The mechanism of energy transfer from an aromatic amino acid to Tb3+ is consistent with a short-range process involving the donor triplet state as described by Dexter (Dexter, D. L. (1953) J. Chem. Phys. 21, 836). This cautions against the use of the Forster equation in approximating distances in these systems

Terbium luminescence in synthetic peptide loops from calcium-binding proteins with different energy donors.

Fourteen 14-mer peptides corresponding to a consensus sequence of metal-binding loops from proteins of the calmodulin family were synthesized. The effect of varying both the position in the binding loop, and the type of aromatic side chains as energy donors for enhancement of terbium luminescence, was studied. It was concluded that tryptophan in loop position 7 gave optimal luminescence enhancement, and that the additional inclusion of a tyrosine in the loop at positions 2 or 4 could further boost emission from the bound terbium. In all further cases energy transfer from aromatic residues at positions other than 7 was markedly less efficient. These results suggest that the peptides assume a configuration which allows a hexadentate ligand structure around the bound terbium ion. This is consistent with a Dexter-type electron exchange model of energy transfer

Structure-Templated Predictions of Novel Protein Interactions from Sequence Information

The multitude of functions performed in the cell are largely controlled by a set of carefully orchestrated protein interactions often facilitated by specific binding of conserved domains in the interacting proteins. Interacting domains commonly exhibit distinct binding specificity to short and conserved recognition peptides called binding profiles. Although many conserved domains are known in nature, only a few have well-characterized binding profiles. Here, we describe a novel predictive method known as domain–motif interactions from structural topology (D-MIST) for elucidating the binding profiles of interacting domains. A set of domains and their corresponding binding profiles were derived from extant protein structures and protein interaction data and then used to predict novel protein interactions in yeast. A number of the predicted interactions were verified experimentally, including new interactions of the mitotic exit network, RNA polymerases, nucleotide metabolism enzymes, and the chaperone complex. These results demonstrate that new protein interactions can be predicted exclusively from sequence information

Reading the face of a leader: women with low facial masculinity are perceived as competitive

In competitive settings, people prefer leaders with masculine faces. But is facial masculinity a trait that is similarly desired in men and women leaders? Across three studies, we discovered that people indeed prefer men and women leaders who have faces with masculine traits. But surprisingly, we find that people also prefer women with low facial masculinity as leaders in competitive contexts (Study 1). Our findings indicate that low facial masculinity in women, but not in men is perceived to indicate competitiveness (Study 2). Thus, in contrast to men, women leaders who rate high in facial masculinity as well as those low in facial masculinity are both selected as leaders in competitive contexts. Indeed, among CEOs of S&P 500 companies, we find a greater range of facial masculinity amongwomen CEOs than among men CEOs (Study 3). Our results suggest that traits of facial masculinity in men and women are interpreted differently. Low facial masculinity in women is linked to competitiveness and not only to cooperativeness as suggested by prior research

The Biomolecular Interaction Network Database and related tools 2005 update

The Biomolecular Interaction Network Database (BIND) (http://bind.ca) archives biomolecular interaction, reaction, complex and pathway information. Our aim is to curate the details about molecular interactions that arise from published experimental research and to provide this information, as well as tools to enable data analysis, freely to researchers worldwide. BIND data are curated into a comprehensive machine-readable archive of computable information and provides users with methods to discover interactions and molecular mechanisms. BIND has worked to develop new methods for visualization that amplify the underlying annotation of genes and proteins to facilitate the study of molecular interaction networks. BIND has maintained an open database policy since its inception in 1999. Data growth has proceeded at a tremendous rate, approaching over 100 000 records. New services provided include a new BIND Query and Submission interface, a Standard Object Access Protocol service and the Small Molecule Interaction Database (http://smid.blueprint.org) that allows users to determine probable small molecule binding sites of new sequences and examine conserved binding residues

Expression of Stretch-Activated Two-Pore Potassium Channels in Human Myometrium in Pregnancy and Labor

Background: We tested the hypothesis that the stretch-activated, four-transmembrane domain, two pore potassium channels (K2P), TREK-1 and TRAAK are gestationally-regulated in human myometrium and contribute to uterine relaxation during pregnancy until labor. Methodology: We determined the gene and protein expression of K2P channels in non-pregnant, pregnant term and preterm laboring myometrium. We employed both molecular biological and functional studies of K2P channels in myometrial samples taken from women undergoing cesarean delivery of a fetus. Principal Findings: TREK-1, but not TREK-2, channels are expressed in human myometrium and significantly up-regulated during pregnancy. Down-regulation of TREK-1 message was seen by Q-PCR in laboring tissues consistent with a role for TREK-1 in maintaining uterine quiescence prior to labor. The TRAAK channel was unregulated in the same women. Blockade of stretch-activated channels with a channel non-specific tarantula toxin (GsMTx-4) or the more specific TREK-1 antagonist L-methionine ethyl ester altered contractile frequency in a dose-dependent manner in pregnant myometrium. Arachidonic acid treatment lowered contractile tension an effect blocked by fluphenazine. Functional studies are consistent with a role for TREK-1 in uterine quiescence. Conclusions: We provide evidence supporting a role for TREK-1 in contributing to uterine quiescence during gestation an

Electric Field Exposure Triggers and Guides Formation of Pseudopod-Like Blebs in U937 Monocytes

We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to regular, round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10-20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb formation. In contrast to round-shaped blebs, PLBs retracted within several minutes after exposure. Cells treated with 1 nM of the actin polymerization blocker cytochalasin D were unable to form PLBs and instead produced stationary, spherical blebs with no elongation or retraction capacity. Live cell fluorescent actin tagging showed that during elongation actin promptly entered the PLB interior, forming bleb cortex and scaffold, which was not seen in stationary blebs. Overall, PLB formation was governed by both passive (physicochemical) effects of membrane permeabilization and active cytoskeleton assembly in the living cell. To a certain extent, PLB mimics the membrane extension in the process of cell migration and can be employed as a nonchemical model for studies of cytomechanics, membrane-cytoskeleton interaction and cell motility

Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells