Yeast phenylalanine transfer RNA: atomic coordinates and torsion angles.

The atomic coordinates of yeast phenylalanine transfer RNA (tRNA) as well as the torsion angles of the polynucleotide chain are presented as derived from an x-ray diffraction analysis of orthorhombic crystals. A comparison is made between the coordinates obtained from analysis of monoclinic crystals of the same material. It is concluded that the molecule has substantially the same form in the orthorhombic and the monoclinic lattices, except for differences found between residues at the 3' end of the polynucleotides chain. A number of observations are made concerning hydrogen bonding interactions which may account for many of the residues conserved in all tRNA sequences.     

A high resolution diffracting crystal form of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase

Three new crystal forms of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase have been produced. The best crystals, obtained after modifying both purification and crystallization conditions, belong to space group P2(1)2(1)2(1) and diffract to 2.7 A. Unit cell parameters are a = 210.4 A, b = 145.3 A and c = 86.0 A (1 A = 0.1 nm), with one dimeric enzyme and two tRNA molecules in the asymmetric unit.     

Preliminary x-ray investigation of an orthorhombic crystal form of human plasma albumin.

An orthorhombic form of single crystals of human plasma albumin, suitable for x-ray diffraction studies, has been grown with ammonium sulfate from protein solutions purified from fresh frozen single donor plasma as well as from a commercial sample of plasma albumin. The space group is P2(1)2(1)2 with 12 molecules in the unit cell. The cell dimensions are: a = 133.3 +/- 1.2 A, b = 274.8 +/- 3.3 A,, and c = 58.02 +/- 0.02 A.     

A new crystal form of ricin-OR

Ricin-OR, an antitumor toxin, has been crystallized in space group P2 with cell parameters a = 8.77 nm, b = 4.64 nm, c = 7.64 nm and beta = 101 degrees. There is one molecule in the asymmetric unit and the solvent content is estimated to be 48% by volume. The crystals diffract to 0.25 nm resolution which is higher than that of the previously reported C2 crystal form which had a solvent content of 65%.     

Yeast mitochondrial transfer RNA. Structure, coding properties and genome organization

The up-to-date data on mitochondrial tRNAs of yeast, their structures and peculiarities of these structures, anomalies of the mitochondrial genetic code and anticodons of tRNAs, the structure and number of tRNA genes are reviewed in the present paper. New information concerning 17 types of yeast mitochondrial tRNAs, deciphered by the authors of the paper are given; among them 8 types are first published. The likeness and differences of yeast mitochondrial tRNAs from their cytoplasmic counterparts are discussed by comparison with other organisms.     

Automated protein crystallization and a new crystal form of a subtilisin:eglin complex

A procedure is described for automating labour-intensive steps of the 'hanging drop' protein crystallization method. An automatic sample changer is employed to fill the wells in a multi-well plate so that concentration gradients in various components are obtained. The sample changer is also used for preparing droplets on a second multi-well plate. Subsequently, this second plate is manually turned around and placed on top of the first multi-well plate such that a large number of chambers with different conditions is obtained simultaneously. During initial trials a new crystal form of a subtilisin:eglin complex was obtained. The crystals have space group P2(1), contain two enzyme inhibitor complexes per asymmetric unit and diffract beyond 2.2 A.     

A new crystal form of tropomyosin. Preliminary X-ray diffraction analysis

A new crystalline form of tropomyosin has been produced that diffracts to about 4 A resolution. The crystals are grown at room temperature by slowly lowering the concentration of spermine. This polyamine apparently neutralizes the acidic amino acid side-chains of tropomyosin and allows close side-by-side packing of molecules. The space group is C2, with unit cell dimensions a = 259.7 A, b = 55.3 A, c = 135.6 A, and beta = 97.2 degrees. The tropomyosin molecules appear to be bonded head-to-tail to form straight filaments that run along the crystallographic (332) direction in an arrangement closely related to thin crystalline sheets previously described.     

Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function

Background  

Despite advances in imaging techniques, real-time visualization of the structure and dynamics of tissues and organs inside small living animals has remained elusive. Recently, we have been using synchrotron x-rays to visualize the internal anatomy of millimeter-sized opaque, living animals. This technique takes advantage of partially-coherent x-rays and diffraction to enable clear visualization of internal soft tissue not viewable via conventional absorption radiography. However, because higher quality images require greater x-ray fluxes, there exists an inherent tradeoff between image quality and tissue damage.     

Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function

Background

Cell shape changes during cytokinesis and chemotaxis require regulation of the actin cytoskeletal network. Dynacortin, an actin cross-linking protein, localizes to the cell cortex and contributes to cortical resistance, thereby helping to define the cell shape changes of cytokinesis. Dynacortin also becomes highly enriched in cortical protrusions, which are sites of new actin assembly.

Results

We studied the effect of dynacortin on cell motility during chemotaxis and on actin dynamics in vivo and in vitro. Dynacortin enriches with the actin, particularly at the leading edge of chemotaxing cells. Cells devoid of dynacortin do not become as polarized as wild-type control cells but move with similar velocities as wild-type cells. In particular, they send out multiple pseudopods that radiate at a broader distribution of angles relative to the chemoattractant gradient. Wild-type cells typically only send out one pseudopod at a time that does not diverge much from 0° on average relative to the gradient. Though dynacortin-deficient cells show normal bulk (whole-cell) actin assembly upon chemoattractant stimulation, dynacortin can promote actin assembly in vitro. By fluorescence spectroscopy, co-sedimentation and transmission electron microscopy, dynacortin acts as an actin scaffolder in which it assembles actin monomers into polymers with a stoichiometry of 1 Dyn₂:1 actin under salt conditions that disfavor polymer assembly.

Conclusion

Dynacortin contributes to cell polarization during chemotaxis. By cross-linking and possibly stabilizing actin polymers, dynacortin also contributes to cortical viscoelasticity, which may be critical for establishing cell polarity. Though not essential for directional sensing or motility, dynacortin is required to establish cell polarity, the third core feature of chemotaxis.     

Studies of interactions of porphyrins with transfer RNA by high-resolution NMR

The interactions of tetra-4N-methylpyridyl porphyrin and its zinc(II), copper(II) and manganese(III) complexes with brewer's yeast type V phenylalanine specific tRNA have been evaluated by high-resolution NMR. Differences in chemical shifts have been noted for three proton resonances in response to the presence of small quantities of the free base and the zinc and copper complexes. The protons giving rise to these signals are located on bases T54 and psi 55, both of which are involved in the primary intraloop and interloop hydrogen bonds that hold the D and T psi C loops together in the tertiary structure. In addition, broadening of specific resonances due to hydrogen bonding protons in the D stem at low ratios of porphyrin to tRNA indicates that the association of porphyrins increases the rate of imino proton exchange. The titration of the tRNA with the manganese(III) complex did not reveal shifts or specific broadening comparable to the other porphyrins at low ratios. The changes induced in the NMR spectrum of tRNA by porphyrins define their site of interaction with the polynucleotide. This site, at the outside of the elbow-bend in the tRNA 'L', is different from the locus of binding in tRNA for other classical DNA intercalators. Furthermore, a new mode of binding may be involved that is neither intercalative nor simply electrostatic.     

New crystal form of cytosolic chicken aspartate aminotransferase suitable for high-resolution X-ray analysis

Four new crystal forms of chicken cytosolic aspartate aminotransferase have been grown from polyethylene glycol solutions. Crystals of the unliganded enzyme and of enzyme liganded with maleate diffract to 1.8 A resolution. Both the free and maleate-liganded enzymes crystallize in space group P2(1)2(1)2(1), but display slightly different cell dimensions (a = 56.9 A, b = 126.9 A and c = 124.6 A versus a = 56.5 A, b = 126.1 A and c = 124.6 A). The influence of various divalent metal ions, dioxane and non-ionic detergent beta-octylglucoside on crystallization has been investigated. The best crystals of liganded enzyme were obtained in the presence of Mg2+ ions, and these crystals were used for data collection to 1.9 A resolution.     

Small-angle x-ray scattering study of lysine transfer RNA ligase from yeast

The scattered X-ray intensities from dilute solutions of lysine transfer RNA ligase, in 0.1 m-phosphate buffer at pH 7.0, have been measured at 21 °. The radius of gyration R (37.5 Å), the molecular weight M (114,000), and the volume V (295,000 ų) were determined.A comparison between the scattering curves obtained from the enzyme and the theoretical scattering curves of different triaxial bodies shows that the shape of the molecule can be represented by an oblate ellipsoid with the semiaxes A = 62.7, B = 50.1 and $\text{C = 23.5}\text{A}\text{?}$.     

A new crystal form of mouse thiamin pyrophosphokinase

Thiamin pyrophosphokinase (TPK) transfers a pyrophosphate group from ATP to the hydroxyl group of thiamin and produces thiamin pyrophosphate (TPP). TPP is the cofactor of metabolically important enzymes such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, transketolase and 2-hydroxyphytanoyl-CoA lyase. Thiamin deficiency results in Wernike-Korsakof Syndrome (WKS) due to neurological disorder and wet beriberi, a potentially fatal cardiovascular disease. Mouse TPK associates as a dimer revealed by previous solved crystallographic structures. In this study, we report mouse TPK complexed with TPP-Mg²⁺ and thiamin -Mg²⁺, respectively, in a new crystal form. In these two structures, four mouse TPK molecules were found in each asymmetric unit. Although we cannot rule out this tetramer form can be an artifact from crystal packing, mouse TPK tetramer has a more closed ATP binding pocket and has the potential to provide specific interactions between mouse TPK and ATP compared with the previous dimeric structure and is likely to be an active form.     

Identification of nucleosides in hydrolysates of transfer RNA by high-resolution mass spectrometry

High-resolution mass spectrometry has been investigated as a technique for identification of modified nucleosides in unfractionated hydrolysates of transfer RNA. The method is based on recognition of predetermined sets of exact mass values which are characteristic of individual nucleoside components. Mass spectra are photographically recorded in a non-time-resolved fashion, so that differing rates of nucleoside vaporization are of no consequence. Experimental parameters of sensitivity, spectrometer resolution and rates of vaporization were studied. Under typical conditions, 1-4 micrograms of tRNA are hydrolyzed, converted to volatile trimethylsilyl derivatives, and mass spectra of the resulting mixture recorded during 3 min at resolution 20 000; 75-100% of the minor nucleosides are usually identified in a single recording. The method is complementary to conventional methods of identification which rely on chromatographic mobilities, and can in principle be generally applied to recognition of biologically or chemically modified bases in nucleic acid.     

The high-resolution crystal structure of porcine pepsinogen.

The structure of porcine pepsinogen at pH 6.1 has been refined to an R-factor of 0.173 for data extending to 1.65 A. The final model contains 180 solvent molecules and lacks density for residues 157-161. The structure of this aspartic proteinase zymogen possesses many of the characteristics of pepsin, the mature enzyme. The secondary structure of the zymogen consists predominantly of beta-sheet, with an approximate 2-fold axis of symmetry. The activation peptide packs into the active site cleft, and the N-terminus (1P-9P) occupies the position of the mature N-terminus (1-9). Thus changes upon activation include excision of the activation peptide and proper relocation of the mature N-terminus. The activation peptide or residues of the displaced mature N-terminus make specific interactions with the substrate binding subsites. The active site of pepsinogen is intact; thus the lack of activity of pepsinogen is not due to a deformation of the active site. Nine ion pairs in pepsinogen may be important in the advent of activation and involve the activation peptide or regions of the mature N-terminus which are relocated in the mature enzyme. The activation peptide-pepsin junction, 44P-1, is characterized by high thermal parameters and weak density, indicating a flexible structure which would be accessible to cleavage. Pepsinogen is an appropriate model for the structures of other zymogens in the aspartic proteinase family.     

Chromatographic fractionation of Escherichia coli transfer RNA on a new support, naphthoyl-Sepharose.

The noncharged naphthoyl-Sepharose CL-6B has been prepared. Escherichia coli tRNA binds to this new adsorbent in 0.75 M ammonium sulphate at neutral pH at room temperature. Using a negative salt gradient, the tRNAs are eluted in a defined order. The chromatographic pattern is clearly different from those of other commonly used tRNA separation techniques.     

Analysis of a new crystal form of procarboxypeptidase B: Further insights into the catalytic mechanism

A new triclinic crystal structure form of porcine pancreatic procarboxypeptidase B (PCPB) was obtained at higher resolution than the previously known tetragonal crystal structure. This new crystal polymorph has allowed for a corrected, accurate assignment of residues along the polypeptide chain based on the currently available gene sequence information and crystallographic data. The present structure shows unbound PCPB in a distinct molecular packing as compared to the previous benzamidine complexed form. Its catalytically important Tyr248 residue is oriented and hydrogen‐bonded to solvent water molecules, and locates the furthest away from the catalytic zinc ion as compared to previous structures. A relatively long stretch of residues flanking Tyr248 and guarding the access to the catalytic zinc ion was found to be sequentially unique to the M14 family of peptidases. Predictions from a normal mode analysis indicated that this stretch of residues belongs to a rigid subdomain in the protein structure. The specific presence of a tyrosyl residue at the most exposed position in this region would allow for a delicate balance between extreme hydrophobicity and hydrophilicity, and affect substrate binding and the kinetic efficiency of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 178–185, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Tyrocidine A: crystal growth and preliminary x-ray diffraction data.

Tyrocidine A was crystallized from 2-methyl-2,4-pentanediol and water, or methanol, to yield crystals that are large enough for X-ray diffraction studies. Four crystals were examined; three were in equilibrium with mother liquor and the fourth was air-dried. They belong to the rhombohedral space group R32. Parameters of the hexagonal cell of the fully solvated crystals vary slightly and are approximately $\text{a = 34}\text{A}\text{?}\text{and}\text{c = 50}\text{A}\text{?}$. The asymmetric unit consists of one molecule of tyrocidine and several molecules of 2-methyl-2,4-pentanediol. Air-dried crystals appear to contain about half the number of solvent molecules. Three-dimensional X-ray diffraction data showing a maximum resolution of $\text{s = (1.7}\text{A}\text{?}\text{)}{}^{\mathrm{?1}}$ have been recorded.