The antifungal protein AFP of Aspergillus giganteus is an oligonucleotide/oligosaccharide binding (OB) fold-containing protein that produces condensation of DNA

The antifungal protein AFP is a small polypeptide of 51 amino acid residues secreted by Aspergillus giganteus. Its potent activity against phytopathogenic fungi converts AFP in a promising tool in plant protection. However, no data have been reported regarding the mode of action of AFP. The three-dimensional structure of this protein, a small and compact beta barrel composed of five highly twisted antiparallel beta strands, displays the characteristic features of the oligonucleotide/oligosaccharide binding (OB fold) structural motif. A comparison of the structures of AFP and OB fold-containing proteins shows this structural similarity despite the absence of any significant sequence similarity. AFP, like most OB fold-containing proteins, binds nucleic acids. The protein promotes charge neutralization and condensation of DNA as demonstrated by electrophoretic mobility shift and ethidium bromide displacement assays. Nucleic acid produces quenching of the protein fluorescence emission. This nucleic acid interacting ability of AFP may be related to the antifungal activity of this small polypeptide.     

The bacterial helicase-primase interaction: a common structural/functional module

The lack of a high-resolution structure for the bacterial helicase-primase complex and the fragmented structural information for the individual proteins have been hindering our detailed understanding of this crucial binary protein interaction. Two new structures for the helicase-interacting domain of the bacterial primases from Escherichia coli and Bacillus stearothermophilus have recently been solved and both revealed a unique and surprising structural similarity to the amino-terminal domain of the helicase itself. In this minireview, the current data are discussed and important new structural and functional aspects of the helicase-primase interaction are highlighted. An attractive structural model with direct biological significance for the function of this complex and also for the development of new antibacterial compounds is examined.     

Lactoferrin and transferrin: functional variations on a common structural framework.

Lactoferrin shares many structural and functional features with serum transferrin, including an ability to bind iron very tightly, but reversibly, a highly-conserved three-dimensional structure, and essentially identical iron-binding sites. Nevertheless, lactoferrin has some unique properties that differentiate it: an ability to retain iron to much lower pH, a positively charged surface, and other surface features that give it additional functions. Here, we review the structural basis for these similarities and differences, including the importance of dynamics and conformational change, and specific interactions that regulate iron binding and release.     

Adrenalectomy and Castration in the Genetically Obese (OB/OB) Mouse

The present studies have tested the hypothesis that adrenalectomy could modify the phenotypic expression of genetic obesity by examining the effects of adrenalectomy on the function of the gonadal system in lean and ob/ob mice. Corticosterone concentrations were undetectable in the adrenalectomized animals. Adrenalectomy significantly slowed the weight gain of obese mice in comparison to sham-adrenalectomized controls. Gonadectomy had no independent effect on weight gain. The testes, prostate, and seminal vesicles in the ob/ob mice were significantly smaller than in the lean animals. Castration lowered the weights of the prostate and seminal vesicles in the lean mice to weights close to those observed in the castrated ob/ob mice. Castration significantly increased the concentrations of LH and FSH in both ob/ob and lean mice, but the absolute concentrations were higher in the lean mice in both conditions. Adrenalectomy per se had no effect on the concentration of LH, FSH, or testosterone or on the weights of the prostate or seminal vesicles. These data indicate that adrenalectomy has no effect on the physiologic control of the reproductive system in genetically obese mice, and are consistent with the hypothesis that the defect in the ob/ob mouse is a modulator of steroid action which over expresses glucocorticoid effects and under expresses gonadal steroid effects.     

Alg mannosyltransferases: From functional and structural analyses to the lipid-linked oligosaccharide pathway reconstitution

BackgroundN-glycosylation is initiated from the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER), which is catalyzed by a series of Alg (asparagine-linked glycosylation) proteins.Scope of reviewThis review summarizes our recent studies on the enzymology of Alg mannosyltransferases (MTases). We also discuss the membrane topology and physiological importance of several ER cytosolic Alg proteins.Major conclusionsUtilizing an efficient prokaryotic protein expression system and a new LC–MS quantitative activity assay, we overexpressed all Alg MTases and performed enzymology studies. Moreover, by reconstituting the LLO pathway, the high-yield chemoenzymatic synthesis of high-mannose-type N-glycans was accomplished using recombinant Alg MTases.General significanceThe analysis of the enzymology and topology of Alg MTases has provided valuable biochemical information in the LLO biosynthesis pathway. In addition, an efficient chemoenzymatic strategy that could prepare various oligomannose-type N-glycans in sufficient amounts was established for further biological assays.     

HspB2/myotonic dystrophy protein kinase binding protein (MKBP) as a novel molecular chaperone: structural and functional aspects

The small heat shock protein, human HspB2, also known as Myotonic Dystrophy Kinase Binding Protein (MKBP), specifically associates with and activates Myotonic Dystrophy Protein Kinase (DMPK), a serine/threonine protein kinase that plays an important role in maintaining muscle structure and function. The structure and function of HspB2 are not well understood. We have cloned and expressed the protein in E.coli and purified it to homogeneity. Far-UV circular dichroic spectrum of the recombinant HspB2 shows a β-sheet structure. Fluorescence spectroscopic studies show that the sole tryptophan residue at the 130(th) position is almost completely solvent-exposed. Bis-ANS binding shows that though HspB2 exhibits accessible hydrophobic surfaces, it is significantly less than that exhibited by another well characterized small HSP, αB-crystallin. Sedimentation velocity measurements show that the protein exhibits concentration-dependent oligomerization. Fluorescence resonance energy transfer study shows that HspB2 oligomers exchange subunits. Interestingly, HspB2 exhibits target protein-dependent chaperone-like activity: it exhibits significant chaperone-like activity towards dithiothreitol (DTT)-induced aggregation of insulin and heat-induced aggregation of alcohol dehydrogenase, but only partially prevents the heat-induced aggregation of citrate synthase, co-precipitating with the target protein. It also significantly prevents the ordered amyloid fibril formation of α-synuclein. Thus, our study, for the first time, provides biophysical characterization on the structural aspects of HspB2, and shows that it exhibits target protein-dependent chaperone-like activity.     

Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs. Exploring the structural diversity by screening for fibroblast growth factor (FGF)1 and FGF2 binding

Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group (3)H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries.     

The hemoglobin of the common sting-ray, Dasyatis sabina: structural and functional properties.

1. The hemoglobin of the sting-ray, Dasyatis sabina, is both polymorphic and heterogeneous; three components predominate. 2. One major component has two kinds of polypeptide chain, of which one, presumably an alpha-chain, has a blocked NH2-terminus and an arginyl COOH-terminus, whereas carboxypeptidases A and B release tyrosine and histidine from the COOH-terminus of the beta-chain. 3. The amino acid sequence of the beginning NH2-terminal segment of the beta-chain of the major component has been determined. 4. The hemoglobin of the sting-ray, Dasyatis sabina, is highly resistant to urea and does not dissociate readily into subunits. 5. Oxygen binding by the hemoglobin is not affected by organic phosphates or high concentrations of either NaCl or urea. 6. The hemoglobin does not polymerize beyond tetramers. 7. Cooperativity, as monitored by n in the Hill equation, is pH-dependent and maximal between pH 8.5 and 9.0. 8. The hemoglobin has a large Bohr effect; the oxygen affinity is 16 times higher at pH 10 than at pH 6.5.     

PROBFIND: a computer program for selecting oligonucleotide probes from peptide sequences.

Synthetic oligonucleotides have proven to be extremely useful probes for screening cDNA and genomic libraries. Selection of the appropriate probe can be more easily and accurately achieved with the use of the computer program PROBFIND. The user enters the amino acid sequence from a file or from the keyboard, selects the minimum length allowed for the probe and the maximum allowable degeneracy. The computer prints a list of the sequences of potential probes which meet these minimum specifications and the location of the corresponding sequence in the protein to the screen and to a file. The user may modify the specifications for length and degeneracy at any time during the output of data, which allows for rapid selection of the desired probe. The program is interactive, accepts any file format with only a single modification of the file, is written in BASIC, and requires less than 6 kbytes of memory. This makes the program easy to use and adaptable even to unsophisticated microcomputers.     

Molecular dynamics simulations of papilloma virus E2 DNA sequences: dynamical models for oligonucleotide structures in solution

The specificity of papilloma virus E2 protein-DNA binding depends critically upon the sequence of a region of the DNA not in direct contact with the protein, and represents one of the simplest known examples of indirect readout. A detailed characterization of this system in solution is important to the further investigation hypothesis of a structural code for DNA recognition by regulatory proteins. In the crystalline state, the E2 DNA oligonucleotide sequence, d(ACCGAATTCGGT), exhibits three different structural forms. We report herein studies of the structure of E2 DNA in solution based on a series of molecular dynamics (MD) simulations including counterions and water, utilizing both the canonical and various crystallographic structures as initial points of departure. All MDs converged on a single dynamical structure of d(ACCGAATTCGGT) in solution. The predicted structure is in close accord with two of the three crystal structures, and indicates that a significant kink in the double helix at the central ApT step in the other crystal molecule may be a packing effect. The dynamical fine structure was analyzed on the basis of helicoidal parameters. The calculated curvature in the sequence was found to originate primarily from YPR steps in the regions flanking the central AATT tract. In order to study the role of structural adaptation of the DNA in the binding process, a subsequent simulation on the 16-mer cognate sequence d(CAACCGAATTCGGTTG) was initiated from the crystallographic coordinates of the bound DNA in the crystal structure of the protein DNA complex. MD simulations starting with the protein-bound form relaxed rapidly back to the dynamical structure predicted from the previous simulations on the uncomplexed DNA. The MD results show that the bound form E2 DNA is a dynamically unstable structure in the absence of protein, and arises as a consequence of both structural changes intrinsic to the sequence and induced by the interaction with protein.     

Studies of cadmium binding to hexokinase: structural and functional implications

The interaction between cadmium and yeast hexokinase was studied. Cadmium produces changes in the aggregation state of the protein and large structures with a large molecular mass were formed. This phenomenon occurs without large modifications to the secondary structure. During this change the enzyme maintains a high level of activity in the monomer as well as in aggregate form. This implies that the enzyme function is not greatly affected by the change and it maintains its active sites without significant modifications. According to kinetic measurements with both glucose and ATP as a variable substrate, cadmium causes a mixed-type inhibition with a main uncompetitive component. Binding experiments show that the protein presents negative cooperative binding with cadmium at various temperatures (298, 303 and 313 K) and a progressive loss in metal union with concentration depending on the temperature. The total union percentage decreases as the metal concentration increases. This is probably due to the aggregation process, which affects the binding sites for the metal and also for the substrate. Labile interactions are more persistent than specific interactions in accordance with the solvation parameter.     

D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.     

Crystal and solution structures of the oligonucleotide d(ATGCGCAT)2: a combined X-ray and NMR study.

A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.     

Biochemical, biophysical, and biological properties of densonucleosis virus (parvovirus). III. common sequences of structural proteins.

Densonucleosis virus cannot code for its four structural proteins if each of them has a unique sequence. The objective of the present investigation, therefore, was to establish whether: (i) the viral genome contains overlapping genes; (ii) the virus incorporates host proteins; or (iii) one of the structural proteins is a dimer. Two independent methods were employed for this purpose. First, the viral proteins, solubilized in sodium dodecyl sulfate, were purified after dansylation and were analyzed by peptide mapping, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, an enzyme-linked immunosorbent assay was developed for a comparative analysis of the viral proteins solubilized by sodium dodecyl sulfate. It was demonstrated with both techniques that densonucleosis virus has four unique structural proteins, all with extensive sequence homologies. Moreover, all structural proteins contained intraprotein, but no interprotein, disulfide linkages. These results indicated similarities between densonucleosis virus and representatives of the two other genera of the Parvoviridae.     

In silico structural and functional modelling of Antifreeze protein (AFP) sequences of Ocean pout (Zoarces americanus, Bloch & Schneider 1801)

Antifreeze proteins (AFPs) are known to polypeptide components formed by certain plants, animals, fungi and bacteria which support to survive in sub-zero temperature. Current study highlighted the seven different antifreeze proteins of fish Ocean pout (Zoarces americanus), in which protein (amino acids sequence) were collected from National Centre for Biotechnology Information and finely characterized using several in silico tools. Such biocomputational techniques applied to figure out the physicochemical, functional and conformational characteristics of targeted AFPs. Multiple physicochemical properties such as Isoelectric Point, Extinction Coefficient and Instability Index, Aliphatic Index, Grand Average Hydropathy were calculated and analysed by ExPASy-ProtParam prediction web server. EMBOSS: pepwheel online tool was used to represent the protein sequences in a helical form. The primary structure analysis shows that most of the AFPs are hydrophobic in nature due to the high content of non-polar residues. The secondary structure of these proteins was calculated using SOPMA tool. SOSUI server and CYS_REC program also run for ideal prediction of transmembrane helices and disulfide bridges of experimental proteins respectively. The modelling of 3D structures of seven desired AFPs were executed by the homology modelling programmes; SWISS MODEL and ProSA web server. UCSF Chimera, Antheprot 3D, PyMOL and RAMPAGE were used to visualize and analysis of the structural variation of the predicted protein model. MEGA7.0.9 software used to know the phylogenetic relationship among these AFPs. These models offered excellent and reliable baseline information for functional characterization of the experimentally derived protein domain composition by using the advanced tools and techniques of Computational Biology.