The conformation of adenovirus VAI-RNA in solution

The secondary structure of an adenovirus associated low molecular weight RNA (VAI-RNA) has been studied by partial digestion with T1-RNase and S1-endonuclease followed by T1-fingerprint analysis. The empirical secondary structure has been compared with two computer generated models based on minimal free energy of the structure. The results suggest that VAI-RNA in solution has a compact structure with a free energy of around -60 kcal with two stems and four bulge regions. The implication of this structure for the function of VAI-RNA is discussed.     

The role of fish parasites in fresh-water ecosystems exemplified by a parasite of the smelt (Osmerus eperlanus)

The European smelt Osmerus eperlanus had been accidentally introduced into the ecosystem of the Syamozero Lake (Karelia). The population of this species has achieved a high density and caused serious changes in the structure and trophic relationships of fish community of the Syamozero ecosystem. The microsporidia Glugea hertwigi Weisenberg, 1921 has become a new and super-dominant parasite of the european smelt in this ecosystem. The invasion of microsporidia has caused a mass death of fishes, that has led to changes in population structure of the smelt and lowered a fish catch. The present study suggests to show a role of parasites in the ichthyocenosis structure regulation in freshwater ecosystem.     

The crystal structure of yeast phenylalanine tRNA at 1.93 A resolution: a classic structure revisited

The crystal structure of the monoclinic form of yeast phenylalanine tRNA has been redetermined at a resolution of 1.93 A. The structure of yeast tRNAphe described here is more accurate than its predecessors not only because it incorporates higher resolution data, but also because it has been refined using techniques that had not been developed when its predecessors were determined more than 20 years ago. The 1.93 A resolution version of this structure differs interestingly from its predecessors in its details. In loop regions particularly, the backbone torsion angles in the new structure are not the same as those reported earlier. Several new divalent cation binding sites have been identified, and the water structure that has emerged is also different.     

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles.

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using 1H-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.     

Isolation and structure of a pseudopeptide gamma-L-glutamyl-L-aspartic acid from Datura stramonium that impairs learning retention in mice

Datura stramonium contains a compound that impairs learning retention in mice. It has been purified to homogeneity and its structure has been established as that of a gamma-L-glutamyl-L-aspartate. The biological activity of this pseudodipeptide has been found to be identical with that of the corresponding synthetic one. It has also been compared to those of various synthetic di- and tripeptides containing L- and/or D-enantiomers of the constitutive amino acids. The results show that the activity is associated with a peptidic structure containing only one type of enantiomer.     

The structure of the amino terminal transforming segment of the p21 protein, Tyr4-Thr20 (with Asp12), by two-dimensional NMR

The structure of a peptide from the transforming region (residues 4-20) of the p21 protein has been determined using two-dimensional NMR. In the normal protein, this segment contains a Gly residue at the critical 12 position; any substitution, other than Pro, at this position results in a transforming protein. Previously performed energy calculations indicated that this peptide segment is a structured one. In this study we find that the Asp12 containing peptide has a surprisingly well-defined structure in solution which has more similarity to the GDP-binding loop region in EF-tu than to that in p21.     

Functional consequences of the excision of an omega loop, residues 40-55, from mitochondrial cytochrome c

A novel technique for protein semisynthesis, enzymic activation, has been used to create a mitochondrial cytochrome c analogue in which the conventional bottom loop has been deleted. The resulting structure resembles that of cytochrome c555 from a primitive photosynthetic sulfur bacteria. Comparisons of this analogue with natural cytochromes show which of the functional differences between cytochromes c and c555 may be related directly to the incorporation of the loop. The structure is an example of an omega loop, recently defined as a discrete category of protein secondary structure. The analogue maintains the overall structure of the parent protein, but a significant change in redox potential has been engineered. It provides support for the prediction that omega loops act as independent modules in folding, function, and evolution. The rapidity of the synthesis and the high yield of product show that this technique for protein engineering is both competitive with, and complementary to, genetic methods.     

Three-dimensional models of HDL apoA-I: implications for its assembly and function

The purpose of this review is to highlight recent advances toward the refinement of a three-dimensional structure for lipid-bound apolipoprotein A-I (apoA-I) on recombinant HDL. Recently, X-ray crystallography has yielded a new structure for full-length, lipid-free apoA-I. Although this approach has not yet been successful in solving the three-dimensional structure of lipid-bound apoA-I, analysis of the X-ray structures has been of immense help in the interpretation of structural data obtained from other methods that yield structural information. Recent studies emphasize the use of mass spectrometry to unambiguously identify cross-linked peptides or to quantify solvent accessibility using hydrogen-deuterium exchange. The combination of mass spectrometry, molecular modeling, molecular dynamic analysis, and small-angle X-ray diffraction has provided additional structural information on apoA-I folding that complements previous approaches.     

Enhanced protein fold recognition using secondary structure information from NMR.

NMR offers the possibility of accurate secondary structure for proteins that would be too large for structure determination. In the absence of an X-ray crystal structure, this information should be useful as an adjunct to protein fold recognition methods based on low resolution force fields. The value of this information has been tested by adding varying amounts of artificial secondary structure data and threading a sequence through a library of candidate folds. Using a literature test set, the threading method alone has only a one-third chance of producing a correct answer among the top ten guesses. With realistic secondary structure information, one can expect a 60-80% chance of finding a homologous structure. The method has then been applied to examples with published estimates of secondary structure. This implementation is completely independent of sequence homology, and sequences are optimally aligned to candidate structures with gaps and insertions allowed. Unlike work using predicted secondary structure, we test the effect of differing amounts of relatively reliable data.     

Novel Dielectric-Loaded Plasmonic Waveguide for Tight-Confined Hybrid Plasmon Mode

In this paper, a novel metal-dielectric waveguide structure is proposed to support hybrid long range surface plasmon polaritons (LRSPPs) with a highly confined mode field. The simulation results showed that our proposed structure has better mode confinement and propagation length compared to that of conventional dielectric-loaded surface plasmon polaritons (DLSPPs) waveguides. This structure offers greater flexibility for the design of surface plasmon polaritons (SPPs) waveguides by altering the trade-off between mode confinement and propagation length. The proposed structure has significant potential for application in highly integrated photonic circuits.     

The structural consequences of exchanging tryptophan and tyrosine residues in B. stearothermophilus lactate dehydrogenase.

A mutant Bacillus stearothermophilus lactate dehydrogenase has been prepared in which all three tryptophan residues in the wild-type enzyme have been replaced by tyrosines. In addition, a tyrosine residue has been mutated to a tryptophan, which acts as a fluorescence probe to monitor protein folding. The mutant enzyme crystallizes in the same crystal form as the wild-type. The crystal structure of the mutant has been determined at 2.8 A resolution. Solution studies have suggested that there is little effect upon the mutant enzyme as judged by its kinetic properties. Comparison of the crystal structures of the mutant and wild-type enzymes confirms this conclusion, and reveals that alterations in structure in the region of these mutations are of a similar magnitude to those observed throughout the structure, and are not significant when compared with the errors in atomic positions expected for a structure at this resolution.     

The new models of the human telomere d[AGGG(TTAGGG)3] in K+ solution

The human telomeric sequence d[AGGG(TTAGGG)(3)] has been found to form different types of G-quadruplex structures. NMR revealed that in Na(+) solution this 22 nucleotide (nt) sequence exhibits an antiparallel structure, whereas crystallographic studies in the presence of K(+) showed a dramatically different parallel structure. The structure of this 22 nt sequence in the presence of K(+) has drawn intense interest as the intracellular K(+) concentration is greater than that of Na(+). However, the question of the type of structure for the 22 nt telomeric sequence in K(+) solution remains open. In this study, we substituted the Gs in the sequence with 8-bromoguanine and examined the resultant structures and thermal stabilities by circular dichroism (CD) spectroscopy. The results suggest that the 22 nt in K(+) solution exists as a mixture of mixed-parallel/antiparallel and chair-type G-quadruplex. To date, the exact structure of human telomeric G-quadruplex in K(+) solution is extremely controversial. The present study provides valuable information for understanding the discrepancies between the crystal and solution studies. We discuss the possible implications of the structure in understanding higher-order telomeric DNA structure and T-loop formation.     

The crystal structure of an ADP complex of Bacillus subtilis pyridoxal kinase provides evidence for the parallel emergence of enzyme activity during evolution

Pyridoxal kinase catalyses the phosphorylation of pyridoxal, pyridoxine and pyridoxamine to their 5' phosphates and plays an important role in the pyridoxal 5' phosphate salvage pathway. The crystal structure of a dimeric pyridoxal kinase from Bacillus subtilis has been solved in complex with ADP to 2.8 A resolution. Analysis of the structure suggests that binding of the nucleotide induces the ordering of two loops, which operate independently to close a flap on the active site. Comparisons with other ribokinase superfamily members reveal that B. subtilis pyridoxal kinase is more closely related in both sequence and structure to the family of HMPP kinases than to other pyridoxal kinases, suggesting that this structure represents the first for a novel family of "HMPP kinase-like" pyridoxal kinases. Moreover this further suggests that this enzyme activity has evolved independently on multiple occasions from within the ribokinase superfamily.     

Crystal structure of a protein associated with cell division from Mycoplasma pneumoniae (GI: 13508053): a novel fold with a conserved sequence motif

UPF0040 is a family of proteins implicated in a cellular function of bacteria cell division. There is no structure information available on protein of this family. We have determined the crystal structure of a protein from Mycoplasma pneumoniae that belongs to this family using X-ray crystallography. Structural homology search reveals that this protein has a novel fold with no significant similarity to any proteins of known three-dimensional structure. The crystal structures of the protein in three different crystal forms reveal that the protein exists as a ring of octamer. The conserved protein residues, including a highly conserved DXXXR motif, are examined on the basis of crystal structure.     

Structural analysis of Saccharomyces cerevisiae myo-inositol phosphate synthase

The New York Structural Genomics Research Consortium has targeted highly conserved but uncharacterized enzyme families for structure determination. As part of this effort, the 2.65-Å crystal structure has been determined for Saccharomyces cerevisiae myo-inositol 1-phosphate synthase (MIP), an essential enzyme that catalyzes critical steps in inositol biosynthesis. The structure determination of four independent monomers in the asymmetric unit (240 kDa) reveals atomic details and residue composition for the partially closed NAD-containing active sites in apo-configuration. The structure further reveals extensive interactions involved in tetrameric assembly of the enzyme complex.     

De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure. Probing the structure at the nanogram level.

The de novo polypeptide named albebetin was designed to form the tertiary fold that has not yet been observed in natural proteins. The design was based on the molecular theory of protein structures. The gene coding for this polypeptide was chemically synthesized. For the initial characterization of a protein structure, a new approach has been developed that uses only nanogram amounts of a polypeptide without its previous purification. This approach includes the biosynthesis of radiolabeled protein in a cell-free translation system with subsequent analysis of its compactness and structure by size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis. According to all tests used, albebetin has a compact stable structure.     

Structural analysis of the lipooligosaccharide from the commensal Haemophilus somnus strain 1P

The structure of the lipooligosaccharide (LOS) from the commensal Haemophilus somnus strain 1P was elucidated. The structure of the O-deacylated LOS was established by monosaccharide analysis, NMR spectroscopy and mass spectrometry. The following structure for the O-deacylated LOS was determined on the basis of the combined data from these experiments. [chemical structure: see text] In the structure Kdo is 3-deoxy-D-manno-octulosonic acid, Hep is L-glycero-D-manno-heptose and lipid A-OH refers to O-deacylated Lipid A. The elucidation of this structure has increased our understanding of the relationship between the variability in LOS structure and the pathogenic potential of this organism. Specifically, the inability of this commensal strain to sialylate its LOS suggests that LOS sialylation could be a crucial virulence factor for H. somnus.     

The secondary structure of myelin basic protein extracted by deoxycholate.

Because of the implication of myelin basic protein in some neurological diseases its in vivo structure is of particular interest. The protein is usually isolated using organic solvents and acid solutions and has previously been shown to contain little alpha-helical or beta-structure; but it is not known how the extraction methods influence the structure. Following recent observations that deoxycholate generally causes minimal structural perturbation when used to dissolve membrane proteins, this detergent has been used to extract the basic protein from bovine myelin. The protein contained in deoxycholate washes of myelin has been purified by gel chromatography and its secondary structure examined by circular dichroism spectroscopy. This protein and conventionally prepared bovine and human basic protein to which 1% deoxycholate has been added appear to have the same structure: they contain 8-14% more helical structure than the chloroform/methanol-extracted protein in pH 4.8 acetate buffer or in pH 9.15 Tris buffer. This conformational change is unaffected by addition of 0.25 M NaCl. The helical content will approach the upper limit if, as is expected, these ordered segments are short. It is suggested that basic protein may adopt this more ordered structure in myelin and possess activity not apparent in its water-soluble unordered conformation. Retention of its encephalitogenic activity following severe treatment may result from an ability to rapidly refold to the original conformation rather than from this activity being inherent in the unordered form.     

Structural organization of the Golgi apparatus

The Golgi apparatus is a universal feature of eukaryotes, carrying out the key functions of processing, sorting and trafficking of newly synthesized membrane and secretory proteins. The Golgi apparatus has a clearly defined structure, comprising stacks of flattened cisternal membranes that in vertebrates are connected to form a ribbon. How this structure is maintained and how it relates to the functions of the Golgi apparatus has long been an area of interest. In this review I describe recent progress in the identification and characterization of the molecular machinery that together help generate the characteristic organization of this organelle.     

Examination of key intermediates in the catalytic cycle of aspartate-beta-semialdehyde dehydrogenase from a gram-positive infectious bacteria

Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes a critical branch point transformation in amino acid bio-synthesis. The products of the aspartate pathway are essential in microorganisms, and this entire pathway is absent in mammals, making this enzyme an attractive target for antibiotic development. The first structure of an ASADH from a Gram-positive bacterium, Streptococcus pneumoniae, has now been determined. The overall structure of the apoenzyme has a similar fold to those of the Gram-negative and archaeal ASADHs but contains some interesting structural variations that can be exploited for inhibitor design. Binding of the coenzyme NADP, as well as a truncated nucleotide analogue, into an alternative conformation from that observed in Gram-negative ASADHs causes an enzyme domain closure that precedes catalysis. The covalent acyl-enzyme intermediate was trapped by soaking the substrate into crystals of the coenzyme complex, and the structure of this elusive intermediate provides detailed insights into the catalytic mechanism.