Correlation between protein stability and crystal properties of designed ROP variants

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.     

Crystal structure of the 28 kDa glutathione S-transferase from Schistosoma haematobium

Schistomiasis is a debilitating parasitic disease which affects 200 million people, causing life-threatening complications in 10% of the patients. This paper reports the crystal structure of the Schistosoma haematobium 28 kDa glutathione S-transferase, a multifunctional enzyme involved in host-parasite interactions and presently considered as a promising vaccine candidate against schistosomiasis. The structures of the GSH-free enzyme, as well as the partially (approximately 40%) and almost fully (approximately 80%) GSH-saturated enzyme, exhibit a unique feature, absent in previous GST structures, concerning the crucial and invariant Tyr10 side chain which occupies two alternative positions. The canonical conformer, which allows an H-bond to be formed between the side chain hydroxyl group and the activated thiolate of GSH, is somewhat less than 50% occupied. The new conformer, with the phenoxyl ring on the opposite side of the mobile loop connecting strand 1 and helix 1, is stabilized by a polar interaction with the guanidinium group of the conserved Arg21 side chain. The presence of two conformers of Tyr10 may provide a clue about clarifying the multiple catalytic functions of Sh28GST and might prove to be relevant for the design of specific antischistosomal drugs. The K(d) for GSH binding was determined by equilibrium fluorescence titrations to be approximately 3 microM and by stopped-flow rapid mixing experiments to be approximately 9 microM. The relatively tight binding of GSH by Sh28GST explains the residually bound GSH in the crystal and supports a possible role of GSH as a tightly bound cofactor involved in the catalytic mechanism for prostaglandin D(2) synthase activity.     

Crystallization and preliminary X-ray analysis of phosphoporin from the outer membrane of Escherichia coli.

Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitable for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A resolution and belong to the space-group P6(3)22, with unit cell dimensions a = b = 121 A and c = 111 A.     

Effect of genome, induction medium and temperature pretreatment on green plant production in durum ( Triticum turgidum var. durum ) x bread wheat ( Triticum aestivum L. em Thell) crosses

The recalcitrancy of durum wheat (Triticum turgidum var. durum) to anther culture, was attempted to be overcome by transferring the responsible genes form bread wheat B-genome to the respective on durum wheat, determining an appropriate induction medium and clarifying the necessity of cold pretreatment. For this, three durum wheat cultivars were crossed to two bread wheat (Triticum aestivum L. em Thell) cultivars. The resulting F₁ plants and their original cultivars were grown in the field and anthers at the appropriate microspore stage were cultured on potato-2 and W₁₄ media with and without low temperature pretreatment. No green plants were produced from the parental durum wheat cultivars. In contrast, green plants were produced from the F₁ plants. The best results in three of the four F₁ hybrids were recorded when potato-2 was used as induction medium. A more variable response of the examined genotypes was noticed with respect to temperature pretreatment. Regarding green plant production, a negative effect of cold pretreatment was observed in two of the F₁ hybrids when they were cultured on potato-2. Chromosome counts on root tips from the resulting green plants revealed that they all carried D-genome chromosomes. The last observation could suggest that D-genome chromosomes are necessary for anther culture response in wheat. Yet, the production of one green plant with 15 chromosomes may indicate that the development of extracted durum genotypes from bread wheat genotypes with good response to in vitro anther culture might be possible. Further work however, is needed for this to be verified.     

A 2.0-A structure of the blue copper protein (cupredoxin) from Alcaligenes faecalis S-6

The structure of a blue copper protein, cupredoxin, from the potent denitrifying bacterium Alcaligenes faecalis S-6, has been determined and refined against 2 A x-ray diffraction data. The agreement between observed and calculated structure factors is 0.159, and estimated errors in coordinates are 0.09-0.15 A. The protein folds in a beta sandwich similar to plastocyanin and azurin and includes features such as a "kink" and a "tyrosine loop" which have been noted previously for these proteins as well as immunoglobulins. The copper is bound by four ligands, in a distorted tetrahedral arrangement, with Cu-S gamma = 2.07 A (Cys-78), Cu-N delta 1 = 2.10 and 2.21 for His-40 and His-81, and Cu-S delta = 2.69 A (Met-86). Two of the ligands are further oriented by hydrogen bonds either to other side chains (Asn-9 to His-40), backbone atoms (NH...S) or a water molecule (to His-40). The methionine ligand has no extra constraints. The C-terminal loop containing three of the ligands is hydrogen-bonded to the strand containing His-40 by hydrogen bonds between the conserved residues Thr-79 and Asn-41. The pronounced dichroism of the crystal is a result of the orientation of the normal to the C beta-S gamma-Cu plane parallel to the crystallographic 6-fold axis.     

Crystallographic phases through genetic engineering: experiences with colicin A

Colicins are antibiotic proteins that kill sensitive Escherichia coli cells. The structure of the pore-forming fragment of colicin A has been solved to 2.5 A resolution using the techniques of X-ray crystallography and genetic engineering. Site-directed mutagenesis was used to construct a number of cysteine-containing mutant proteins, one of which yielded an excellent mercurial derivative. Our experiences suggest strategies for obtaining useful heavy-atom derivatives for protein crystallography using genetic engineering techniques.