Protein crystallization for genomics: throughput versus output

Structural genomics involves many steps in order to reach from Gene to structure. This article focuses on the crystallization step in this chain of tasks. It is becoming increasingly evident that the current high throughput procedures for crystallising proteins do not always produce the expected output of high quality crystals required for structure determination by x-ray crystallography. This problem is discussed and suggestions for raising the output are presented.     

Optimal strategy for structured model of fishing problem

In this work we study a structured fishing model, basically displaying the two stages of the ages of a fish population, which are in our case juvenile, and adults. We associate to this model the maximization of the total discounted net revenues derived by the exploitation of the stock. The exploitation strategy of the optimal control problem is then developed and presented.     

Role of calcium as trigger in thermal beta-lactoglobulin aggregation

Divalent calcium ions have been suggested to be involved in intermolecular protein-Ca2+-protein cross-linking, intramolecular electrostatic shielding, or ion-induced protein conformational changes as a trigger for protein aggregation at elevated temperatures. To address the first two phenomena in the case of beta-lactoglobulin, a combination of chemical protein modification, calcium-binding, and aggregation studies was used, while the structural integrity of the modified proteins was maintained. Although increasing the number of carboxylates on the protein by succinylation results in improved calcium-binding, calcium appears to be less effective in inducing protein aggregation. In fact, the larger the number of carboxylates, the higher the concentration of calcium that is required to trigger the aggregation. Lowering the number of negative charges on the protein surface via methylation of carboxylates reduces calcium-binding properties, but calcium-induced aggregation at low concentration is improved. Monovalent sodium ions cannot take over the specific role of calcium. The relation between net surface charge and number of calcium ions bound required to trigger the aggregation suggests that calcium needs to bind site specific to carboxylates with a threshold affinity. Subsequent site-specific screening of surface charges results in protein aggregation, driven by the partial unfolding of the protein at elevated temperatures, which is then facilitated by the absence of electrostatic repulsion.     

Sulfated and pyruvylated disaccharide alditols obtained from a red seaweed galactan: ESIMS and NMR approaches

The water-soluble acid agaran isolated from Acanthophora spicifera (Rhodophyta) was submitted to alkaline treatment for the complete cyclization of alpha-L-Galp 6-sulfate to 3,6-An-alpha-L-Galp units. The modified agaran was then partially depolymerized using partial reductive hydrolysis. The resulting oligosaccharide mixture was fractionated by adsorption and ion-exchange chromatography. Fractions were purified by gel-filtration chromatography and studied by ESIMS and NMR spectroscopy, including 1D 1H, 13C, DEPT and 2D 1H, 1H COSY, TOCSY and 1H, 13C HMQC procedures. The following neutral, pyruvylated, sulfated and sulfated/pyruvylated disaccharide alditols were obtained: beta-D-Galp-(1-->4)-3,6-An-L-GalOH; 4,6-O-(1-carboxyethylidene)-beta-D-Galp-(1-->4)-3,6-An-L-GalOH; beta-D-Galp 2-sulfate-(1-->4)-3,6-An-L-GalOH and 4,6-O-(1-carboxyethylidene)-beta-D-Galp 2-sulfate-(1-->4)-3,6-An-L-GalOH.     

Structural biology of mammalian lipoxygenases: enzymatic consequences of targeted alterations of the protein structure

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the pathogenesis of diseases with major health political relevance (bronchial asthma, atherosclerosis, cancer, and osteoporosis). The crystal structures of one mammalian lipoxygenase and of two plant isoenzymes have been solved and the structural bases of important enzyme properties (reaction specificity, membrane binding, and suicidal inactivation) have been investigated in the past. This review will briefly summarize our current understanding on the structural biology of the most important mammalian lipoxygenase isoforms and will also address selected mechanistic features of the lipoxygenase reaction.     

Theoretical studies of Alzheimer's disease drug candidate 3-[(2,4-dimethoxy)benzylidene]-anabaseine (GTS-21) and its derivatives

Theoretical and molecular modeling studies have been conducted for understanding the details of how 3-[(2,4-dimethoxy)benzylidene]-anabaseine dihydrochloride (GTS-21) and its metabolism derivatives bind with the receptor of alpha7 nicotinic acetylcholine dimer. Good accordance with experimental results has been achieved. It was found that the van der Waals repulsion makes the dominant contribution to the binding energy. GTS-21 and its metabolites are apparently too large for the binding sites of the alpha7 dimer. To improve the effectiveness of the drug, a possible approach is to reduce its volume while maintaining the presence of the active groups. Our studies, in combination with experimental studies, will lead to a promising basis for practical drug design against Alzheimer's disease.     

Glyoxalase I from Leishmania donovani: a potential target for anti-parasite drug

Glyoxalases are involved in a ubiquitous detoxification pathway. In pursuit of a better understanding of the biological function of the enzyme, the recombinant glyoxalase I (LdGLOI) protein has been characterized from Leishmania donovani, the most important pathogenic Leishmania species that is responsible for visceral leishmaniasis. A 24kDa protein was heterologously expressed in Escherichia coli. LdGLOI showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOI protein could detect a band of anticipated size approximately 16kDa in promastigote extracts. Several inhibitors of human GLOI showed that they are weak inhibitors of L. donovani growth. Overexpression of GLOI gene in L. donovani using Leishmania expression vector pspalpha hygroalpha, we detected elevated expression of GLOI RNA and protein. Comparative modelling of the 3-D structure of LDGLOI shows that substrate-binding region of the model involves important differences compared to the homologues, such as E. coli, specific to glutathione. Most notably a substrate-binding loop of LDGLOI is characterized by a deletion of five residues compared to the E. coli homologue. Further, a critical Arg in the E. coli variant at the substrate-binding site is replaced by Tyr in LDGLOI. These major differences result in entirely different shapes of the substrate-binding loop and presence of very different chemical groups in the substrate-binding site of LDGLOI compared to E. coli homologue suggesting an explanation for the difference in the substrate specificity. Difference in the substrate specificity of the human and LDGLOI enzyme could be exploited for structure-based drug designing of selective inhibitors against the parasite.     

The structure of kappa/iota-hybrid carrageenans II. Coil-helix transition as a function of chain composition

This paper describes the effect of the kappa/iota-ratio on the physical properties of kappa/iota-hybrid carrageenans (synonyms: kappa-2, kappa-2, weak kappa, weak gelling kappa). To this end, a series of kappa/iota-hybrid carrageenans ranging from almost homopolymeric kappa-carrageenan (98 mol-% kappa-units) to almost homopolymeric-carrageenan (99 mol-% iota-units) have been extracted from selected species of marine red algae (Rhodophyta). The kappa/iota-ratio of these kappa/iota-hybrids was determined by NMR spectroscopy. Their rheological properties were determined by small deformation oscillatory rheology. The gel strength (storage modulus, G') of the kappa/iota-hybrids decreases with decreasing kappa-content. On the other hand, the gelation temperature of the kappa-rich kappa/iota-hybrids is independent of their composition. This allows one to control the gel strength independent of the gelation or melting temperature. The conformational order-disorder transition of the kappa/iota-hybrids was studied using optical rotation and high-sensitivity differential scanning calorimetry. High-sensitivity DSC showed that the total transition enthalpy of the kappa/iota-hybrids goes through a minimum at 60 mol-% kappa-units, whereas for the mixture of kappa- and iota-carrageenan, the total transition enthalpy is a linear function of the composition. With respect to the ordering capability, the kappa/iota-hybrid carrageenans seem to behave as random block copolymers with length sequence distributions truncated from the side of the small lengths. Intrinsic thermodynamic properties (e.g., transition temperature and enthalpy) of kappa- and iota-sequences in these copolymers are close to those of their parent homopolymers. The critical sequence length for kappa-sequences is 2-fold of that for iota-sequences.     

Insight into the self-association of key enzymes from pathogenic species

Self-association of protein monomers to higher-order oligomers plays an important role in a plethora of biological phenomena. The classical biophysical technique of analytical ultracentrifugation is a key method used to measure protein oligomerisation. Recent advances in sedimentation data analysis have enabled the effects of diffusion to be deconvoluted from sample heterogeneity, permitting the direct identification of oligomeric species in self-associating systems. Two such systems are described and reviewed in this study. First, we examine the enzyme dihydrodipicolinate synthase (DHDPS), which crystallises as a tetramer. Wild-type DHDPS plays a critical role in lysine biosynthesis in microbes and is therefore an important antibiotic target. To confirm the state of association of DHDPS in solution, we employed sedimentation velocity and sedimentation equilibrium studies in an analytical ultracentrifuge to show that DHDPS exists in a slow dimer–tetramer equilibrium with a dissociation constant of 76 nM. Second, we review works describing the hexamerisation of GDP-mannose pyrophosphorylase (GDP-MP), an enzyme that plays a critical role in mannose metabolism in Leishmania species. Although the structure of the GDP-MP hexamer has not yet been determined, we describe a three-dimensional model of the hexamer based largely on homology with the uridyltransferase enzyme, Glmu. GDP-MP is a novel drug target for the treatment of leishmaniasis, a devastating parasitic disease that infects more than 12 million people worldwide. Given that both GDP-MP and DHDPS are only active in their oligomeric states, we propose that inhibition of the self-association of critical enzymes in disease is an emerging paradigm for therapeutic intervention.     

Structural and nucleotide-binding properties of YajQ and YnaF,two Escherichia coli proteins of unknown function

Structural genomics is a new approach in functional assignment of proteins identified via whole-genome sequencing programs. Its rationale is that nonhomologous proteins performing similar or related biological functions might have similar tertiary structure. We used dye pseudoaffinity chromatography, two-dimensional gel electrophoresis, and mass spectrometry to identify two novel Escherichia coli nucleotide-binding proteins, YnaF and YajQ. YnaF exhibited significant sequence identity with MJ0577, an ATP-binding protein from a hyperthermophile (Methanococcus jannaschii), and with UspA, a protein from Haemophilus influenzae that belongs to the Universal Stress Protein family. YnaF conserves the ATP-binding site and the dimeric structure observed in the crystal of MJ0577. The protein YajQ, present in many bacterial genomes, is missing in eukaryotes. In the absence of significant similarities of YajQ to any solved structure, we determined its structural and ligand-binding properties by NMR and isothermal titration calorimetry. We demonstrate that YajQ is composed of two domains, each centered on a beta-sheet, that are connected by two helical segments. NMR studies, corroborated with local sequence conservation among YajQ homologs in various bacteria, indicate that one of the beta-sheets is mostly involved in biological activity.