Crystal structure of atypical cytoplasmic ABC-ATPase SufC from Thermus thermophilus HB8

SufC, a cytoplasmic ABC-ATPase, is one of the most conserved Suf proteins. SufC forms a stable complex with SufB and SufD, and the SufBCD complex interacts with other Suf proteins in the Fe-S cluster assembly. We have determined the crystal structure of SufC from Thermus thermophilus HB8 in nucleotide-free and ADP-Mg-bound states at 1.7A and 1.9A resolution, respectively. The overall architecture of the SufC structure is similar to other ABC ATPases structures, but there are several specific motifs in SufC. Three residues following the end of the Walker B motif form a novel 3(10) helix which is not observed in other ABC ATPases. Due to the novel 3(10) helix, a conserved glutamate residue involved in ATP hydrolysis is flipped out. Although this unusual conformation is unfavorable for ATP hydrolysis, salt-bridges formed by conserved residues and a strong hydrogen-bonding network around the novel 3(10) helix suggest that the novel 3(10) helix of SufC is a rigid conserved motif. Compared to other ABC-ATPase structures, a significant displacement occurs at a linker region between the ABC alpha/beta domain and the alpha-helical domain. The linker conformation is stabilized by a hydrophobic interaction between conserved residues around the Q loop. The molecular surfaces of SufC and the C-terminal helices of SufD (PDB code: 1VH4) suggest that the unusual linker conformation conserved among SufC proteins is probably suitable for interacting with SufB and SufD.     

Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants

The 2.5 A crystal structure of Escherichia coli glutaminyl-tRNA synthetase in a quaternary complex with tRNA(Gln), an ATP analog and glutamate reveals that the non-cognate amino acid adopts a distinct binding mode within the active site cleft. In contrast to the binding of cognate glutamine, one oxygen of the charged glutamate carboxylate group makes a direct ion-pair interaction with the strictly conserved Arg30 residue located in the first half of the dinucleotide fold domain. The nucleophilic alpha-carboxylate moiety of glutamate is mispositioned with respect to both the ATP alpha-phosphate and terminal tRNA ribose groups, suggesting that a component of amino acid discrimination resides at the catalytic step of the reaction. Further, the other side-chain carboxylate oxygen of glutamate is found in a position identical to that previously proposed to be occupied by the NH(2) group of the cognate glutamine substrate. At this position, the glutamate oxygen accepts hydrogen bonds from the hydroxyl moiety of Tyr211 and a water molecule. These findings demonstrate that amino acid specificity by GlnRS cannot arise from hydrogen bonds donated by the cognate glutamine amide to these same moieties, as previously suggested. Instead, Arg30 functions as a negative determinant to drive binding of non-cognate glutamate into a non-productive orientation. The poorly differentiated cognate amino acid-binding site in GlnRS may be a consequence of the late emergence of this enzyme from the eukaryotic lineage of glutamyl-tRNA synthetases.     

Sterics and solvation winnow accessible conformational space for unfolded proteins

The magnitude of protein conformational space is over-estimated by the traditional random-coil model, in which local steric restrictions arise exclusively from interactions between adjacent chain neighbors. Using a five-state model, we assessed the extent to which steric hindrance and hydrogen bond satisfaction, energetically significant factors, impose additional conformational restrictions on polypeptide chains, beyond adjacent residues. Steric hindrance is repulsive: the distance of closest approach between any two atoms cannot be less than the sum of their van der Waals radii. Hydrogen bond satisfaction is attractive: polar backbone atoms must form hydrogen bonds, either intramolecularly or to solvent water. To gauge the impact of these two factors on the magnitude of conformational space, we systematically enumerated and classified the disfavored conformations that restrict short polyalanyl backbone chains. Applying such restrictions to longer chains, we derived a scaling law to estimate conformational restriction as a function of chain length. Disfavored conformations predicted by the model were tested against experimentally determined structures in the coil library, a non-helix, non-strand subset of the PDB. These disfavored conformations are usually absent from the coil library, and exceptions can be uniformly rationalized.     

Near-Infrared Analysis of Hydrogen-Bonding in Glass- and Rubber-State Amorphous Saccharide Solids

Near-infrared (NIR) spectroscopic analysis of noncrystalline polyols and saccharides (e.g., glycerol, sorbitol, maltitol, glucose, sucrose, maltose) was performed at different temperatures (30–80°C) to elucidate the effect of glass transition on molecular interaction. Transmission NIR spectra (4,000–12,000 cm⁻¹) of the liquids and cooled-melt amorphous solids showed broad absorption bands that indicate random configuration of molecules. Heating of the samples decreased an intermolecular hydrogen-bonding OH vibration band intensity (6,200–6,500 cm⁻¹) with a concomitant increase in a free and intramolecular hydrogen-bonding OH group band (6,600–7,100 cm⁻¹). Large reduction of the intermolecular hydrogen-bonding band intensity at temperatures above the glass transition (T _g) of the individual solids should explain the higher molecular mobility and lower viscosity in the rubber state. Mixing of the polyols with a high T _g saccharide (maltose) or an inorganic salt (sodium tetraborate) shifted both the glass transition and the inflection point of the hydrogen-bonding band intensity to higher temperatures. The implications of these results for pharmaceutical formulation design and process monitoring (PAT) are discussed.     

Production of a Firm,Resilient Gel-forming Polysaccharide by a Mutant of Alcaligenes faecalis var. myxogenes 10C3

Isolation and purification procedures of yeast pro-proteinase C are described. In order to obtain a good yield of the proenzyme, it was necessary to repeat the inactivating treatment of coexisting yeast proteinase A and to perform purification procedures as rapidly as possible at low temperature. The purified protein was homogeneous with respect to chromatographic, electrophoretic and sedimentation criteria. The proenzyme possessed no inherent activity for acetyl-l-tyrosine ethylester but a slight activation seemed to occur during the activity assay. The molecular weight of the proenzyme was 79,200 as determined by the sedimentation and diffusion methods. Its other physicochemical properties were compared with those of proteinase C. The proenzyme as well as proteinase C was shown to be a glycoprotein which contains 8~12% true sugars.     

Synthesis and structural characterization of enantiopure levodropropizine

Enantiopure levodropropizine can be synthesized in high yield by the direct nucleophilic addition of 1-phenylpiperazine to (R)-glycidol followed by recrystallization in absolute ethanol. A chiral HPLC method has been developed for the determination of its optical purity. The enantiomeric enrichment behavior of this compound in the recrystallization process is discussed according to its binary melting point phase diagram. Single crystal x-ray structural analysis shows the intermolecular hydrogen-bonding interactions between the hydroxyl and piperazinyl groups within the crystal lattice of levodropropizine. © 1996 Wiley-Liss, Inc.     

π-Facial hydrogen bonding in the chiral resolving agent 2,2,2-trifluoro-1-(9-anthryl)-ethanol and its racemic modification

2,2,2-Trifluoro-(9-anthryl)-ethanol (TFAE) has been extensively used, in its pure enantiomeric forms, as a chiral solvating agent in nuclear magnetic resonance spectroscopy (NMR). It has also played an important role in the development of chiral stationary phases in liquid chromatography (LC). X-ray crystallography of the enantiomeric and racemic crystals shows, in both cases, the formation of an intermolecular hydrogen bond between the O? H and the π-face of one of the rings of the anthracene aromatic system.¹ Few examples of such hydrogen bonding have been published previously, and those that have are not as clear cut as in this case. An explanation for the hydrogen bonding is sought using molecular modelling via the PM3 analytically derived molecular electrostatic potentials. Using NMR and dynamic lineshape analysis, the barrier to rotation about the aryl-carbon bond is estimated, indicating the C? CF₃ bond to be perpendicular to the anthracene axis in nonpolar solution. This conformation is identical to the conformation in the crystal. Evidence is also presented to support the formation of intermolecular π-facial hydrogen bonding in TFAE solutions. It is thought that such hydrogen bonding may be implicated in chiral recognition using this compound. © 1994 Wiley-Liss, Inc.