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The characterization of crystal defects induced by irradiation, such as X-rays, charged particles and neutrons, is important for understanding radiation damage and the associated generation of defects. Radiation damage to protein crystals has been measured using various methods. Until now, these methods have focused on decreased diffraction intensity, volume expansion of unit cells and specific damage to side chains. Here, the direct observation of specific crystal defects, such as dislocations, induced by X-ray irradiation of protein crystals at room temperature is reported. Dislocations are induced even by low absorbed doses of X-ray irradiation. This study revealed that for the same total absorbed dose, the formation of defects appears to critically depend on the dose rate. The relationship between dislocation energy and dose energy was analyzed based on dislocation theory associated with elasticity theory for crystalline materials. This demonstration of the crystal defects induced by X-ray irradiation could help to understand the underlying mechanisms of X-ray-induced radiation damage.

Protein encapsulation has long attracted many chemists and biologists because of its potential to control the structure and functions of proteins, but has been a daunting challenge because of their incommensurably larger size compared with common synthetic hosts. Here we report the encapsulation of a small protein, ubiquitin, within giant coordination cages. The protein was attached to one bidentate ligand and, upon addition of Pd(II) ions (M) and additional ligands (L), M(12)L(24) coordination nanocages self-assembled around the protein. Because of the well-defined host framework, the protein-encapsulated structure could be analysed by NMR spectroscopy, ultracentrifugation and X-ray crystallography.

Background: Deer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant. Results: Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules. Conclusions: The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1β2/α2β1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites. [ABSTRACT FROM AUTHOR]

The features of hydrogen bonds in DsrD protein from sulfate-reducing bacteria have been investigated by neutron protein crystallography. The function of DsrD has not yet been elucidated clearly, but its X-ray crystal structure revealed that it comprises a winged-helix motif and shows the highest structural homology to the DNA-binding proteins. Since any neutron structure of a DNA recognition protein has not yet been obtained, here detailed information on the hydrogen bonds in the winged-helix-motif protein is given and the following features found. (i) The number of hydrogen bonds per amino acid of DsrD is relatively fewer than for other proteins for which neutron structures were determined previously. (ii) Hydrogen bonds are localized between main-chain and main-chain atoms; there are few hydrogen bonds between main-chain and side-chain atoms and between side-chain and side-chain atoms. (iii) Hydrogen bonds inducted by protonation of specific amino acid residues (Glu50) seem to play an essential role in the dimerization of DsrD. The former two points are related to the function of the DNA-binding protein; the three-dimensional structure was mainly constructed by hydrogen bonds in main chains, while the side chains appeared to be used for another role. The latter point would be expected to contribute to the crystal growth of DsrD. [ABSTRACT FROM AUTHOR]

Studies hydration structures in proteins and neutron diffraction experiment on dissimilatory sulte reductase D. Water molecules adopting a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen-bond formation and dynamics of hydration; Hydration patterns observed in neutron Fourier maps.

Dissimilatory sulfite reductase D (DsrD) from Desulfovibrio vulgaris has been crystallized for u neutron diffraction study. The initial crystals obtained were too small for the neutron experiment. In order to obtain a larger crystal (>1 mm[SUP4]), a combination of two techniques was developed to determine the optimum crystallization conditions: a crystallization phase diagram was obtained, followed by crystal quality assessment via X-ray diffraction. Using conditions determined in this manner, a large single crystal (1.7 mm[SUP3]) of DsrD protein was subsequently grown in D[SUB2]O solution by the macroseeding technique, A neutron diffraction experiment was carried out using the BIX-3 diffractometer at the Japan Atomic Energy Research Institute (JAERI). collecting data to 2.4 Å resolution from an optimized crystal. [ABSTRACT FROM AUTHOR]

6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII′) that has only ∼0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII′ proteins; EII′-level activity) by x-ray crystallography at 1.8 Å resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of D-alanyl-D-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for D-alanyl-D-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, D-alanyl-D-alanine-peptidase, and β-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser112 as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp181 and Asn266) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the β-lactamase fold resulted in the evolution of the nylon oligomer hydrolase. [ABSTRACT FROM AUTHOR]