Ring inversion barrier of diazepam and derivatives: An ab Initio study

Systematic ab initio calculations were performed to investigate the ring inversion process of various 1,4‐diazepines including diazepam, N¹‐desmethyldiazepam, and 3‐methyl‐N¹‐desmethyldiazepam. The diazepine ring adopts a shape of a boat; owing to asymmetric substitution two such boats are possible in mirror image relation to each other. In the present study both structural and solvent effects were investigated on the energetics of ring inversion of nine diazepine derivatives. The calculated ring inversion barriers for diazepam (17.6 kcal/mol) and N¹‐desmethyldiazepam (10.9 kcal/mol) are in good agreement with the corresponding experimental data. In the cases of diazepam and N¹‐desmethyldiazepam, the calculated minimum energy path of the ring inversion is asymmetric contrary to the fact that the terminals (M and P conformers) are equienergetic. Chirality 11:651–658, 1999. © 1999 Wiley‐Liss, Inc.     

Oxygen Adsorption on Zr(0001): An ab Initio Study

Abstract

The main goal of this paper is to study the oxygen adsorption on the Zr(0001) surface using first-principles total-energy calculations based on density-functional theory. We present preliminary results which concern the atomic oxygen adsorption on the Zr(0001) surface. We first report a static study where we calculate the atomic structure and adsorption energy for oxygen occupying various surface and subsurface sites at three different coverages: Θ = 1/4, 1/2, and 1 ML. We find that oxygen atoms are preferentially adsorbed into the octahedral holes between the 2nd and 3rd metallic layers. We secondly perform ab initio molecular dynamics calculations for the Zr(0001) – (3 × 3) – O system to show how the oxygen can penetrate through the surface and how it finally reaches its equilibrium position, trapped between the 1st and 2nd zirconium layers.     

Integration of Evolutionary Features for the Identification of Functionally Important Residues in Major Facilitator Superfamily Transporters

The identification of functionally important residues is an important challenge for understanding the molecular mechanisms of proteins. Membrane protein transporters operate two-state allosteric conformational changes using functionally important cooperative residues that mediate long-range communication from the substrate binding site to the translocation pathway. In this study, we identified functionally important cooperative residues of membrane protein transporters by integrating sequence conservation and co-evolutionary information. A newly derived evolutionary feature, the co-evolutionary coupling number, was introduced to measure the connectivity of co-evolving residue pairs and was integrated with the sequence conservation score. We tested this method on three Major Facilitator Superfamily (MFS) transporters, LacY, GlpT, and EmrD. MFS transporters are an important family of membrane protein transporters, which utilize diverse substrates, catalyze different modes of transport using unique combinations of functional residues, and have enough characterized functional residues to validate the performance of our method. We found that the conserved cores of evolutionarily coupled residues are involved in specific substrate recognition and translocation of MFS transporters. Furthermore, a subset of the residues forms an interaction network connecting functional sites in the protein structure. We also confirmed that our method is effective on other membrane protein transporters. Our results provide insight into the location of functional residues important for the molecular mechanisms of membrane protein transporters.     

An Approach to Identifying the Functionally Important RNA Sites by Complementary Addressed Modification

An approach based on complementary addressed modification of nucleic acids by oligodeoxyribonucleotide derivatives was proposed for changing the spatial structure of particular RNA sites in order to study their role in the biological activity of the total RNA molecule. Hepatitis C virus (HCV) IRES was used as a model. Oligodeoxyribonucleotide derivatives contained a 4-[N-(2-chloroethyl)-N-methylamino]benzylamino group at the 5-phosphate and were complementary to various RNA sites located in regions of hairpins II, IIId, or IIIe. Covalent adducts resulting from RNA alkylation with the derivatives were isolated by denaturing PAGE and tested for binding with the 40S subunit of human ribosomes. Structural alteration of hairpin II had no effect on the binding, whereas that of hairpin IIIe substantially reduced it. The RNA with modified hairpin IIId showed virtually no binding with the 40S subunit. Hairpin IIId was assumed to play the critical role in the binding of HCV IRES with the 40S subunit.     

Functional Consequences of Changing Proline Residues in the Phenylalanine-Specific Permease of Escherichia coli

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on genetic analysis involving the construction of protein fusions with alkaline phosphatase has previously been proposed in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm (J. Pi and A. J. Pittard, J. Bacteriol. 178:2650–2655, 1996). Site-directed mutagenesis has been used to investigate the functional importance of each of the 16 proline residues of the PheP protein. Replacement of alanine at only three positions, P54, P341, and P442, resulted in the loss of 50% or more activity. Substitutions at P341 had the most dramatic effects. None of these changes in transport activity were, however, associated with any defect of the mutant protein in inserting into the membrane, as indicated by [³⁵S]methionine labelling and immunoprecipitation using anti-PheP serum. A possible role for each of these three prolines is discussed. Inserting a single alanine residue at different sites within span IX and the loop immediately preceding it also had major effects on transport activity, suggesting an important role for a highly organized structure in this region of the protein.     

Phylogenetic Gaussian Process Model for the Inference of Functionally Important Regions in Protein Tertiary Structures

A critical question in biology is the identification of functionally important amino acid sites in proteins. Because functionally important sites are under stronger purifying selection, site-specific substitution rates tend to be lower than usual at these sites. A large number of phylogenetic models have been developed to estimate site-specific substitution rates in proteins and the extraordinarily low substitution rates have been used as evidence of function. Most of the existing tools, e.g. Rate4Site, assume that site-specific substitution rates are independent across sites. However, site-specific substitution rates may be strongly correlated in the protein tertiary structure, since functionally important sites tend to be clustered together to form functional patches. We have developed a new model, GP4Rate, which incorporates the Gaussian process model with the standard phylogenetic model to identify slowly evolved regions in protein tertiary structures. GP4Rate uses the Gaussian process to define a nonparametric prior distribution of site-specific substitution rates, which naturally captures the spatial correlation of substitution rates. Simulations suggest that GP4Rate can potentially estimate site-specific substitution rates with a much higher accuracy than Rate4Site and tends to report slowly evolved regions rather than individual sites. In addition, GP4Rate can estimate the strength of the spatial correlation of substitution rates from the data. By applying GP4Rate to a set of mammalian B7-1 genes, we found a highly conserved region which coincides with experimental evidence. GP4Rate may be a useful tool for the in silico prediction of functionally important regions in the proteins with known structures.     

An Excess-Calcium-Binding-Site Model Predicts Neurotransmitter Release at the Neuromuscular Junction

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca²⁺-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20–40 independent Ca²⁺-binding sites on synaptic vesicles, only a fraction of which need to bind Ca²⁺ to trigger fusion, are sufficient to predict physiological release. Our excess-Ca²⁺-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca²⁺-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca²⁺ dynamics during synapse activation.     

An Excess-Calcium-Binding-Site Model Predicts Neurotransmitter Release at the Neuromuscular Junction

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca²⁺-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20–40 independent Ca²⁺-binding sites on synaptic vesicles, only a fraction of which need to bind Ca²⁺ to trigger fusion, are sufficient to predict physiological release. Our excess-Ca²⁺-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca²⁺-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca²⁺ dynamics during synapse activation.     

Identification of Functionally Important Residues of the Silkmoth Pheromone Biosynthesis-activating Neuropeptide Receptor,an Insect Ortholog of the Vertebrate Neuromedin U Receptor

The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by the interaction between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class A G-protein-coupled receptor. To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions was generated using a PBANR chimera C-terminally fused with enhanced GFP. The PBANR mutants were expressed in Sf9 insect cells, and their ability to bind and be activated by a core PBAN fragment (C10PBAN^R2K) was monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, whereas the other four remained intracellular. Reduced binding relative to wild type was observed with 17 mutants, and decreased Ca²⁺ mobilization responses were observed with 12 mutants. Ala substitution of Glu-95, Glu-120, Asn-124, Val-195, Phe-276, Trp-280, Phe-283, Arg-287, Tyr-307, Thr-311, and Phe-319 affected both binding and Ca²⁺ mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the 2nd, 3rd, 6th, and 7th transmembrane helices, implying that the same general region of class A G-protein-coupled receptors recognizes both peptidic and nonpeptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair, neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, Glu-95 and Glu-120, in silkmoth PBANR and Glu-117 and Glu-142 in human NMUR1, in the recognition of the most functionally critical region of the ligands, the C-terminal residue and amide.     

A Suppressor Analysis of Residues Involved in Cation Transport in the Lactose Permease: Identification of a Coupling Sensor

Four amino acids critical for lactose permease function were altered using site-directed mutagenesis. The resulting Quad mutant (E269Q/R302L/H322Q/E325Q) was expressed at 60% of wild-type levels but found to have negligible transport activity. The Quad mutant was used as a parental strain to isolate suppressors that regained the ability to ferment the α-galactoside melibiose. Six different suppressors were identified involving five discrete amino acid changes and one amino acid deletion (Q60L, V229G, Y236D, S306L, K319N and ΔI298). All of the suppressors transported α-galactosides at substantial rates. In addition, the Q60L, ΔI298 and K319N suppressors regained a small but detectable amount of lactose transport. Assays of sugar-driven cation transport showed that both the Q60L and K319N suppressors couple the influx of melibiose with cations (H⁺ or H₃O⁺). Taken together, the data show that the cation-binding domain in the lactose permease is not a fixed structure as proposed in previous models. Rather, the data are consistent with a model in which several ionizable residues form a dynamic coupling sensor that also may interact directly with the cation and lactose.     

Search for Functionally Significant Motifs and Amino Acid Residues of Actin

The scientific interest to the structural and functional properties of actin is determined by its abundance in cells. Being an important component of the cytoskeleton, actin is involved in many protein-protein interactions. Using crystal structures and molecular models, we have mapped the amino acid residues that are involved in these interactions and form the ATP-binding site of the actin monomer. Moreover, using mass spectrometry and high-performance liquid chromatography methods, we have discovered the regions of the amino acid sequence of actin that form the core of the actin fibril. According to the bioinformatic analysis, these regions are amyloidogenic and are located in the C-terminal region and in the hinge between the first and third subdomains. The data obtained are applicable to chordate actin, because multiple alignment revealed highly conserved amino acid sequences. In turn, the comparison of the chordate actin with the bacterial homologs showed the presence of numerous amino acid substitutions and insertions.     

Prediction of Functionally Important Phospho-Regulatory Events in Xenopus laevis Oocytes

The African clawed frog Xenopus laevis is an important model organism for studies in developmental and cell biology, including cell-signaling. However, our knowledge of X. laevis protein post-translational modifications remains scarce. Here, we used a mass spectrometry-based approach to survey the phosphoproteome of this species, compiling a list of 2636 phosphosites. We used structural information and phosphoproteomic data for 13 other species in order to predict functionally important phospho-regulatory events. We found that the degree of conservation of phosphosites across species is predictive of sites with known molecular function. In addition, we predicted kinase-protein interactions for a set of cell-cycle kinases across all species. The degree of conservation of kinase-protein interactions was found to be predictive of functionally relevant regulatory interactions. Finally, using comparative protein structure models, we find that phosphosites within structured domains tend to be located at positions with high conformational flexibility. Our analysis suggests that a small class of phosphosites occurs in positions that have the potential to regulate protein conformation.     

Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)

Transporters essential for neurotransmission in mammalian organisms and bacterial multidrug transporters involved in antibiotic resistance are evolutionarily related. To understand in more detail the evolutionary aspects of the transformation of a bacterial multidrug transporter to a mammalian neurotransporter and to learn about mechanisms in a milieu amenable for structural and biochemical studies, we identified, cloned, and partially characterized bacterial homologues of the rat vesicular monoamine transporter (rVMAT2). We performed preliminary biochemical characterization of one of them, Brevibacillus brevis monoamine transporter (BbMAT), from the bacterium B. brevis. BbMAT shares substrates with rVMAT2 and transports them in exchange with >1H⁺, like the mammalian transporter. Here we present a homology model of BbMAT that has the standard major facilitator superfamily fold; that is, with two domains of six transmembrane helices each, related by 2-fold pseudosymmetry whose axis runs normal to the membrane and between the two halves. The model predicts that four carboxyl residues, a histidine, and an arginine are located in the transmembrane segments. We show here that two of the carboxyls are conserved, equivalent to the corresponding ones in rVMAT2, and are essential for H⁺-coupled transport. We conclude that BbMAT provides an excellent experimental paradigm for the study of its mammalian counterparts and bacterial multidrug transporters.     

Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors

We have combined alanine mutagenesis and functional assays to identify amino acid residues in the channel domain that are critical for inositol 1,4,5-trisphosphate receptor (IP₃R) channel function. The residues selected were highly conserved in all three IP₃R isoforms and were located in the cytosolic end of the S6 pore-lining helix and proximal portion of the C-tail. Two adjacent hydrophobic amino acids (Ile-2588 and Ile-2589) at the putative cytosolic interface of the S6 helix inactivated channel function and could be candidates for the channel gate. Of five negatively charged residues mutated, none completely eliminated channel function. Of five positively charged residues mutated, only one inactivated the channel (Arg-2596). In addition to the previously identified role of a pair of cysteines in the C-tail (Cys-2610 and Cys-2613), a pair of highly conserved histidines (His-2630 and His-2635) were also essential for channel function. Expression of the H2630A and H2635A mutants (but not R2596A) produced receptors with destabilized interactions between the N-terminal fragment and the channel domain. A previously unrecognized association between the cytosolic C-tail and the TM 4,5-loop was demonstrated using GST pulldown assays. However, none of the mutations in the C-tail interfered with this interaction or altered the ability of the C-tail to assemble into dimers. Our present findings and recent information on IP₃R structure from electron microscopy and crystallography are incorporated into a revised model of channel gating.     

Functionally Important Residues in the Predicted 3<Superscript>rd</Superscript> Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)

The type IIa Na⁺/P_i, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na⁺ and one divalent P_i ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and P_i transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of P_i transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the P_i-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na⁺-dependent P_i transport. Since electrogenic NaPi-II transports 3 Na⁺/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na⁺ binding sites in NaPi-IIa.     

Mutagenic mechanism of the A-T to G-C transition induced by 5-bromouracil: an ab Initio Study

The tautomerisms of uracil, 5-bromouracil (BrU), G-U, G-BrU, A-U, and A-BrU have been studied theoretically in an effort to investigate the mutagenicity of BrU. The ab initio calculations have been performed using HF and B3LYP methods with various basis sets. The relative stability of all tautomers was established. The intermolecular interactions between U, BrU, U*, BrU* (asterisks denote enol forms), and water have been studied. It shows that the possibility of tautomerism from BrU to BrU* is much more likely than that from U to U*. Further research indicates that BrU* tends to pair with guanine more easily than U*. The proton transfer process has been investigated by potential energy surface (PES) scan and transition state analysis. The results show that the proton transfer between G-U* and G*-U is monodirectional barrier-free proton transfer (BFPT), which terminates the base mismatch induced by U*. On the other hand, the proton transfer between G-BrU* and G*-BrU is bidirectional BFPT, which makes the base mismatch induced by BrU* sustained. On the basis of all of these calculated results, a new mutagenic mechanism for the A-T to G-C transition induced by 5-bromouracil is described in detail for the first time. It might give a new insight into the origin of the mutagenicity of the 5-Br derivative.     

Mutational Analysis of the Candida albicans Ammonium Permease Mep2p Reveals Residues Required for Ammonium Transport and Signaling

The ammonium permease Mep2p mediates ammonium uptake and also induces filamentous growth in the human-pathogenic yeast Candida albicans in response to nitrogen limitation. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is not required for ammonium transport but is essential for Mep2p-dependent morphogenesis. Progressive C-terminal truncations showed Y433 to be the last amino acid that is essential for the induction of filamentous growth, thereby delimiting the Mep2p signaling domain. To understand in more detail how the signaling activity of Mep2p is regulated by ammonium availability and transport, we mutated conserved amino acid residues that have been implicated in ammonium binding or uptake. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is thought to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filament formation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filament formation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filament formation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. These results provide important insights into ammonium transport and control of morphogenesis by Mep2p in C. albicans.     

Thermal and Hydrodynamic Environments Mediate Individual and Aggregative Feeding of a Functionally Important Omnivore in Reef Communities

In eastern Canada, the destruction of kelp beds by dense aggregations (fronts) of the omnivorous green sea urchin, Strongylocentrotus droebachiensis, is a key determinant of the structure and dynamics of shallow reef communities. Recent studies suggest that hydrodynamic forces, but not sea temperature, determine the strength of urchin-kelp interactions, which deviates from the tenets of the metabolic theory of ecology (MTE). We tested the hypothesis that water temperature can predict short-term kelp bed destruction by S. droebachiensis in calm hydrodynamic environments. Specifically, we experimentally determined relationships among water temperature, body size, and individual feeding in the absence of waves, as well as among wave velocity, season, and aggregative feeding. We quantified variation in kelp-bed boundary dynamics, sea temperature, and wave height over three months at one subtidal site in Newfoundland to test the validity of thermal tipping ranges and regression equations derived from laboratory results. Consistent with the MTE, individual feeding during early summer (June-July) obeyed a non-linear, size- and temperature-dependent relationship: feeding in large urchins was consistently highest and positively correlated with temperature <12°C and dropped within and above the 12–15°C tipping range. This relationship was more apparent in large than small urchins. Observed and expected rates of kelp loss based on sea temperature and urchin density and size structure at the front were highly correlated and differed by one order of magnitude. The present study speaks to the importance of considering body size and natural variation in sea temperature in studies of urchin-kelp interactions. It provides the first compelling evidence that sea temperature, and not only hydrodynamic forces, can predict kelp bed destruction by urchin fronts in shallow reef communities. Studying urchin-seaweed-predator interactions within the conceptual foundations of the MTE holds high potential for improving capacity to predict and manage shifts in marine food web structure and productivity.