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Terahertz (THz) resonance absorption originates from intermolecular interactions, which are suitable for identifying amino acids with multiple isomers. L-threonine and L-allo-threonine are diastereomers with two characteristic peaks in the effective spectrum range of 1.0-2.3 THz, which are located at 1.42 and 2.14 THz for L-threonine (L-thr) and 1.63 and 2.16 THz for L-allo-threonine (L-allo-thr). Based on the density functional theory (DFT) of the crystal structures of L-thr and L-allo-thr, the vibration frequencies of 1.56, 1.87, 2.16 THz, and 2.22 THz were obtained, corresponding to the THz characteristic peaks. Through vibration model analysis, it was found that lattice and skeleton vibrations mediated by intermolecular hydrogen bonds play a crucial role in the THz response. Studying the experimental absorption spectra of different proportions co-crystallized mixtures and 1:1 physical mixture of L-thr and L-allo-thr, it was found that the characteristic peaks of the physical mixture include the characteristic peaks of the two diastereomers in the THz band, while amino-acid co-crystallized mixtures formed their own characteristic peaks depending on the proportion. The results show that the co-crystallized mixture composition of diastereomers can be quantitatively analyzed by THz time-domain spectroscopy (THz-TDS)., As one of the eight essential proteinogenic amino acids, L-threonine (L-thr) is required for body growth, uric acid metabolism, and the immune system. L-allo-threonine (L-allo-thr) is used as a starting [...]

The preferences of amino acid residues for [phi], [psi] backbone angles vary strikingly among the amino acids, as shown by the backbone angle [phi] found from the [sup.3]J([H.sub.[alpha]], [H.sub.N]) coupling constant for short peptides in water. New data for the [sup.3]([H.sub.[alpha]], [H.sub.N]) values of blocked amino acids (dipeptides) are given here. Dipeptides exhibit the full range of coupling constants shown by longer peptides such as GGXGG and dipeptides present the simplest system for analyzing backbone preferences. The dipeptide coupling constants are surprisingly close to values computed from the coil library (conformations of residues not in helices and not in sheets). Published coupling constants for GGXGG peptides agree closely with dipeptide values for all nonpolar residues and for some polar residues but not for X = D, N, T, and Y, which are probably affected by polar side chain-backbone interactions in GGXGG peptides. Thus, intrinsic backbone preferences are already determined at the dipeptide level and remain almost unchanged in GGXGG peptides and are strikingly similar in the coil library of conformations from protein structures. The simplest explanation for the backbone preferences is that backbone conformations are strongly affected by electrostatic dipole-dipole interactions in the peptide backbone and by screening of these interactions with water, which depends on nearby side chains. Strong backbone electrostatic interactions occur in dipeptides. This is shown by calculations both of backbone electrostatic energy for different conformers of the alanine dipeptide in the gas phase and by electrostatic solvation free energies of amino acid dipeptides. amino acid conformations | dipeptides | electrostatic screening

We present a detailed structural characterization of the intermediate state populated during the folding and unfolding of the bacterial immunity protein Im7. We achieve this result by incorporating a variety of experimental data available for this species in molecular dynamics simulations. First, we define the structure of the exchange-competent intermediate state of Im7 by using equilibrium hydrogen-exchange protection factors. Second, we use this ensemble to predict [PHI]-values and compare the results with the experimentally determined [PHI]-values of the kinetic refolding intermediate. Third, we predict chemical-shift measurements and compare them with the measured chemical shifts of a mutational variant of Im7 for which the kinetic folding intermediate is the most stable state populated at equilibrium. Remarkably, we found that the properties of the latter two species are predicted with high accuracy from the exchange-competent intermediate that we determined, suggesting that these three states are characterized by a similar architecture in which helices I, II, and IV are aligned in a native-like, but reorganized, manner. Furthermore, the structural ensemble that we obtained enabled us to rationalize the results of tryptophan fluorescence experiments in the WT protein and a series of mutational variants. The results show that the integration of diverse sets of experimental data at relatively low structural resolution is a powerful approach that can provide insights into the structural organization of this conformationally heterogeneous three-helix intermediate with unprecedented detail and highlight the importance of both native and non-native interactions in stabilizing its structure. hydrogen exchange | protein folding | protein intermediates

Abstract: This research integrated the Revised Universal Soil Loss Equation (RUSLE) and the soil statistical analysis with RS and GIS techniques to quantify soil and nutrients loss risk. The Letianxi [...]

The Mars Organic Analyzer (MOA), a microfabricated capillary electrophoresis (CE) instrument for sensitive amino acid biomarker analysis, has been developed and evaluated. The microdevice consists of a four-wafer sandwich combining glass CE separation channels, microfabricated pneumatic membrane valves and pumps, and a nanoliter fluidic network. The portable MOA instrument integrates high voltage CE power supplies, pneumatic controls, and fluorescence detection optics necessary for field operation. The amino acid concentration sensitivities range from micromolar to 0.1 nM, corresponding to part-per-trillion sensitivity. The MOA was first used in the lab to analyze soil extracts from the Atacama Desert, Chile, detecting amino acids ranging from 10-600 parts per billion. Field tests of the MOA in the Panoche Valley, CA, successfully detected amino acids at 70 parts per trillion to 100 parts per billion in jarosite, a sulfate-rich mineral associated with liquid water that was recently detected on Mars. These results demonstrate the feasibility of using the MOA to perform sensitive in situ amino acid biomarker analysis on soil samples representative of a Mars-like environment. amino acid analysis | astrobiology | capillary electrophoresis | microfabrication

The related marine natural products halichlorine, pinnaic acid, and tauropinnaic acid have been synthesized. The described route provided access to all three compounds from a common, late-stage intermediate. The synthesis began with 1-pyrrolidino-1-cyclopentene from which an intermediate possessing the three contiguous stereocenters of the natural products was synthesized in just four steps. Olefin cross metathesis followed by a hydrogenation/hydrogenolysis reaction stereoselectively formed the piperidine ring. Use of a [beta]-lactam group provided internal protection for the highly congested nitrogen atom during side-chain elaboration. The [beta]-lactam was subsequently reduced directly to an amino aldehyde, which after the Horner-Wadsworth-Emmons reaction was elaborated to pinnaic acid. The same amino aldehyde was also transformed into halichlorine after a thiol-mediated cyclization sequence to form the dehydroquinolizidine ring system.

The Directional Atomic Solvation Energy (DASEY) is an atom-based description of the environment of an amino acid position within a known 3D protein structure. The DASEY has been developed to align and score a probe amino acid sequence to a library of template protein structures for fold assignment. DASEY is computed by summing the atomic solvation parameters of atoms falling within a tetrahedral sector, or petal, extending 16 [Angstrom] along each of the four bond axes of each [alpha]-carbon atom of the protein. The DASEY discriminates between pairs of structurally equivalent positions and random pairs in protein structures sharing a fold but belonging to different superfamilies, unlike some previous descriptors of protein environments, such as buried area. Furthermore, the DASEY values have characteristic patterns of residue replacement, an essential feature of a successful fold assignment method. Benchmarking fold assignment with DASEY achieves coverage of 56% of sequences with 90% accuracy when probe sequences are matched to protein structural templates belonging to the same fold but to a different superfamily, an improvement of greater than 200% over a previous method.

There is a paradox concerning the [beta] propensities of the amino acids: the amino acids with the highest [beta] propensities such as valine and isoleucine have the highest tendency to desolvate the peptide backbone, which should result in a loss of stability. Nevertheless, backbone solvation, calculated as electrostatic solvation free energy (ESF), is highly correlated with mutant stability in the zinc-finger system studied by Kim and Berg [Kim, C. A. & Berg, J. M. (1993) Nature (London) 362, 267-270], and valine and isoleucine are among the most stabilizing amino acids. This inverse correlation between stability and ESF can be explained, because the mutant [DELTA]ESF differences in the unfolded protein are larger than in the native protein. Consequently, mutations such as Ala to Val destabilize the unfolded form more than the native protein. By comparing mutant [DELTA]ESF values in isolated [beta]-strands versus [beta]-sheets, we conclude that amino acids with high [beta] propensities should exert their stabilizing effects at early stages in folding. This deduction agrees with the studies by Clarke and coworkers [Lorch, M., Mason, J. M., Clarke, A. R. & Parker, M. J. (1999) Biochemistry 38, 1377-1385, and Lorch, M., Mason, J. M., Sessions, R. B. & Clarke, A. R. (2000) Biochemistry 39, 3480-3485] of the thermodynamics of folding of the [beta]-sheet protein CD2.d1.

Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. E[C.sub.50]s for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the E[C.sub.50] for L-glutamate to activate the anion conductance, without affecting the E[C.sub.50] for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.

Conjugation in Blepharisma japonicum is induced by interaction between complementary mating-types I and II, which excrete blepharmone (gamone 1) and blepharismone (gamone 2), respectively. Gamone 1 transforms type II cells such that they can unite, and gamone 2 similarly transforms type I cells. Moreover, each gamone promotes the production of the other gamone. Gamone 2 has been identified as calcium-3-(2'-formylamino-5'-hydroxybenzoyl) lactate and has been synthesized chemically. Gamone 1 was isolated and characterized as a glycoprotein of 20-30 kDa containing 175 amino acids and 6 sugars. However, the amino acid sequence and arrangement of sugars in this gamone are still unknown. To determine partial amino acid sequences of gamone 1, we established a method of isolation based on the finding that this glycoprotein can be concentrated by a Con A affinity column. Gamone 1 is extremely unstable and loses its biological activity once adsorbed to any of the columns that we tested. By using a Con A affinity column and native PAGE, we detected a 30-kDa protein corresponding to gamone I activity and determined the partial amino acid sequences of the four peptides. To isolate gamone 1 cDNA, we isolated mRNA from mating-type I cells stimulated by synthetic gamone 2 and then performed rapid amplification of cDNA ends procedures by using gene-specific primers and cloned cDNA of gamone 1. The cDNA sequence contains an ORF of 305 amino acids and codes a possibly novel protein. We also estimated the arrangement of sugars by comparing the affinity to various lectin columns.

[beta]-Lactamases are the resistance enzymes for [beta]-lactam antibiotics, of which four classes are known. [beta]-lactamases hydrolyze the [beta]-lactam moieties of these antibiotics, rendering them inactive. It is shown herein that the class D OXA-10 [beta]-lactamase depends critically on an unusual carbamylated lysine as the basic residue for both the enzyme acylation and deacylation steps of catalysis. The formation of carbamylated lysine is reversible. Evidence is presented that this enzyme is dimeric and carbamylated in living bacteria. High-resolution x-ray structures for the native enzyme were determined at pH values of 6.0, 6.5, 7.5, and 8.5. Two dimers are present per asymmetric unit. One monomer in each dimer was carbamylated at pH 6.0, whereas all four monomers were fully carbamylated at pH 8.5. At the intermediate pH values, one monomer of each dimer was carbamylated, and the other showed a mixture of carbamylated and non-carbamylated lysines. It would appear that, as the pH increased for the sample, additional lysines were 'titrated' by carbamylation. A handful of carbamylated lysines are known from protein crystallographic data, all of which have been attributed roles in structural stabilization (mostly as metal ligands) of the proteins. This paper reports a previously unrecognized role for a noncoordinated carbamylate lysine as a basic residue involved in mechanistic reactions of an enzyme, which indicates another means for expansion of the catalytic capabilities of the amino acids in nature beyond the 20 common amino acids in development of biological catalysts.

Drosophila Enabled (Ena) was first identified as a genetic suppressor of mutations in the Abelson tyrosine kinase and subsequently was shown to be a member of the Ena/vasodilator-stimulated phosphoprotein family of proteins. All members of this family have a conserved domain organization, bind the focal adhesion protein zyxin, and localize to focal adhesions and stress fibers. Members of this family are thought to be involved in the regulation of cytoskeleton dynamics. The Ena protein sequence has multiple poly(L-proline) residues with similarity to both profilin and src homology 3 binding sites. Here, we show that Ena can bind directly to the Drosophila homolog of profilin, chickadee. Furthermore, Ena and profilin were colocalized in spreading cultured cells. We report that the proline-rich region of Ena is responsible for this interaction as well as for mediating binding to the src homology 3 domain of the Abelson tyrosine kinase. These data support the hypothesis that Ena provides a regulated link between signal transduction and cytoskeleton assembly in the developing Drosophila embryo.

The FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) and [p70.sup.s6k] (the 70-kDa S6 kinase). Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and [p70.sup.s6k] in response to mitogenic stimuli. We present evidence that FRAP controls 4E-BP1 and [p70.sup.s6k] phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of [p70.sup.s6k], and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A is shown to associate with [p70.sup.s6k] but not with a mutated [p70.sup.s6k] that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A in vitro, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BPI and [p70.sup.s6k], whereas amino acid deprivation or rapamycin treatment inhibits FRAP's ability to restrain the phosphatase.

GATA transcription factors are divided into two subfamilies that play key roles in hematopoiesis and cardiogenesis. GATA-4 is an important regulator expressed in overlapping patterns in the heart, urogenital system and smooth muscle lineages. A FOG-related protein, FOG-2 has been observed to be coexpressed with GATA-4 in the brain and tissues of the urogenital system and also in both developing and adult heart. The interactions of FOG-2 with the N-terminal zinc finger of GATA-4 and its repression of GATA-4's transcriptional activity in the heart show its potential importance.

Protein aggregation is studied by following the simultaneous folding of two designed identical 20-letter amino acid chains within the framework of a lattice model and using Monte Carlo simulations. It is found that protein aggregation is determined by elementary structures (partially folded intermediates) controlled by local contacts among some of the most strongly interacting amino acids and formed at an early stage in the folding process.

In the maximum parsimony (MP) and minimum evolution (ME) methods of phylogenetic inference, evolutionary trees are constructed by searching for the topology that shows the minimum number of mutational changes required (M) and the smallest sum of branch lengths (S), respectively, whereas in the maximum likelihood (ML) method the topology showing the highest maximum likelihood (A) of observing a given data set is chosen. However, the theoretical basis of the optimization principle remains unclear. We therefore examined the relationships of M, S, and A for the MP, ME, and ML trees with those for the true tree by using computer simulation. The results show that M and S are generally greater for the true tree than for the MP and ME trees when the number of nucleotides examined (n) is relatively small, whereas A is generally lower for the true tree than for the ML tree. This finding indicates that the optimization principle tends to give incorrect topologies when n is small. To deal with this disturbing property of the optimization principle, we suggest that more attention should be given to testing the statistical reliability of an estimated tree rather than to finding the optimal tree with excessive efforts. When a reliability test is conducted, simplified MP, ME, and ML algorithms such as the neighbor-joining method generally give conclusions about phylogenetic inference very similar to those obtained by the more extensive tree search algorithms.

We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering experiments allow the definition of motions in the nano- to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic atomic fluctuation regime in the global behavior of proteins. Biological activity in proteins is correlated with this transition, suggesting that flexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR containing H-retinal, H-tryptophan, and H-methionine was prepared biosynthetically in Halobacterium salinarum. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temperature, the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These experimental results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global flexibility), on the one hand, to allow large conformational changes in the molecule and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.

The [Alpha]- and [Beta]-karyopherins (Kaps), also called importins, mediate the nuclear transport of proteins. All [Alpha]-Kaps contain a central domain composed of eight approximately 40 amino acid, tandemly arranged, armadillo-like (Arm) repeats. The number and order of these repeats have not changed since the common origin of fungi, plants, and mammals. Phylogenetic analysis suggests that the various [Alpha]-Kaps fall into two groups, [Alpha]1 and [Alpha]2. Whereas animals encode both types, the yeast genome encodes only an [Alpha]1-Kap. The [Beta]-Kaps are characterized by 14-15 tandemly arranged HEAT motifs. We show that the Arm repeats of [Alpha]-Kaps and the HEAT motifs of [Beta]-Kaps are similar, suggesting that the [Alpha]-Kaps and [Beta]-Kaps (and for that matter, all Arm and HEAT repeat-containing proteins) are members of the same protein superfamily. Phylogenetic analysis indicates that there are at least three major groups of [Beta]-Kaps, consistent with their proposed cargo specificities. We present a model in which an [Alpha]-independent [Beta]-Kap progenitor gave rise to the [Alpha]-dependent [Beta]-Kaps and the [Alpha]-Kaps.