ABSTRACT
Molecular and electronic structures for four p-carborane derivatives were studied in the context of their liquid crystalline properties. Thus molecular and crystal structures of diheptyl and diheptynyl derivatives of 10- and 12-vertex bi-p-carboranes were determined by X-ray crystallography and compared to the results of ab initio calculations at the HF/6-31G level of theory. Experimentally observed significant positional disorder of one of the substituents in the 10-vertex derivatives, 2[2]a and 2[2]b, was related to conformational properties of the alkyl-carborane bond. Experimental and theoretical studies of the electronic structures were conducted for the four compounds using UV and NMR spectroscopies. The nature of the unique long wavelength absorption band at 232 nm in the diheptynyl derivative 2[2]b was explained using INDO/2//HF/6-31G analysis. The complete assignment of the (13)C signals was accomplished using a long-range coupling technique and was supported by the calculated (HF/6-31G) isotropic shielding tensors. Analysis of absorption spectra, NMR substituent effects, and trends in bond lengths shows generally strong cage-acetylene electronic interactions for the 10-vertex p-carborane, while the 12-vertex p-carborane remains largely electronically isolated. Ab initio calculations revealed that 12-vertex p-carborane has significantly larger electronic polarizability and quadrupole moments than the 10-vertex analogues, which are larger than those for bicyclo[2.2.2]octane compounds. All these results on packing, conformational, and electronic properties form the basis for the discussion of thermal behavior of the four carborane compounds, bicyclo[2.2.2]octane analogues, and some related compounds.
ABSTRACT
The three-dimensional X-ray crystal structure of full-length oxidized bovine adrenodoxin (Adx) has been determined at 2.5 A resolution by molecular replacement using a structure of a truncated form as a starting model. Crystals of Adx belong to a primitive monoclinic space group P2(1) with four Adx molecules in an asymmetric unit. The unit cell dimensions are a = 59.44 A, b = 77.03 A, c = 59.68 A, and beta = 94.83 degrees. The structure has been refined to an R factor of 23.5%. Structures of the four molecules of full-length Adx (127 amino acids) in the asymmetric unit were compared with each other and also with that of the truncated Adx (4-108). The overall topology of full-length Adx remains the same as described earlier for the truncated protein. Differences that do occur are almost wholly confined to alternate side-chain conformations that reflect differing lattice contacts made by two proteins. Extensive interactions found between molecules 1 and 2 in the full-length Adx asymmetric unit may reflect the ability of Adx to form dimers in vivo and are consistent with hydrodynamic measurements which show that in solution there is an equilibrium between monomeric and dimeric forms of Adx. Dimerization of Adx could explain why the truncated form has greater affinity for the P450 redox partner than the full-length form. From these results it can be considered that the mechanism of electron transfer is not necessarily the same in different mitochondrial P450 systems.
Subject(s)
Adrenodoxin/chemistry , Adrenodoxin/genetics , Adrenodoxin/metabolism , Animals , Catalytic Domain , Cattle , Crystallization , Crystallography, X-Ray , Cytochrome P-450 Enzyme System/metabolism , Dimerization , Electrochemistry , Electron Transport , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SolutionsABSTRACT
The structure of the tetradeca-(3-fluorotyrosyl) M1-1 GSH transferase (3-FTyr GSH transferase), a protein in which tyrosine residues are globally substituted by 3-fluorotyrosines has been determined at 2.2 A resolution. This variant was produced to study the effect on the enzymatic mechanism and the structure was undertaken to assess how the presence of the 3-fluorotyrosyl residue influences the protein conformation and hence its function. Although fluorinated amino acid residues have frequently been used in biochemical and NMR investigations of proteins, no structure of a protein that has been globally substituted with a fluorinated amino acid has previously been reported. Thus, this structure represents the first crystal structure of such a protein containing a library of 14 (28 crystallographically distinct) microenvironments from which the nature of the interactions of fluorine atoms with the rest of the protein can be evaluated. Numerous conformational changes are observed in the protein structure as a result of substitution of 3-fluorotyrosine for tyrosine. The results of the comparison of the crystal structure of the fluorinated protein with the native enzyme reveal that conformational changes are observed for most of the 3-fluorotyrosines. The largest differences are seen for residues where the fluorine, the OH, or both are directly involved in interactions with other regions of the protein or with a symmetry-related molecule. The fluorine atoms of the 3-fluorotyrosine interact primarily through hydrogen bonds with other residues and water molecules. In several cases, the conformation of a 3-fluorotyrosine is different in one of the monomers of the enzyme from that observed in the other, including different hydrogen-bonding patterns. Altered conformations can be related to differences in the crystal packing interactions of the two monomers in the asymmetric unit. The fluorine atom on the active-site Tyr6 is located near the S atom of the thioether product (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene and creates a different pattern of interactions between 3-fluorotyrosine 6 and the S atom. Studies of these interactions help explain why 3-FTyr GSH transferase exhibits spectral and kinetic properties distinct from the native GSH transferase.
Subject(s)
Crystallography, X-Ray/methods , Glutathione Transferase/chemistry , Protein Conformation , Tyrosine/analogs & derivatives , Animals , Binding Sites , Dimerization , Hydrogen Bonding , Models, Molecular , Rats , Tyrosine/chemistry , Water/chemistry , Xenobiotics/chemistryABSTRACT
It has been suggested that the muscles of the anterolateral abdominal wall increase the stability of the lumbar region of the vertebral column by tensing the thoracolumbar fascia and by raising intra-abdominal pressure. In this report these new mechanisms are reviewed and their contribution to vertebral stability assessed. The thoracolumbar fascia consists of two principal layers of dense fibrous tissue that attach the abdominal muscles to the vertebral column. Each of these layers was dissected in fresh and fixed material and samples chosen for light and scanning electron microscopy to study the arrangement of the component fibers. Computed axial tomography in volunteers showed the changes in spatial organization that occur during flexion of the back and during the Valsalva maneuver. The fascia was then tensed experimentally in isolated unfixed motion segments. The results suggested that the stabilizing action of the thoracolumbar fascia is less than had been thought previously but was consistent with calculations based on the more accurate structural and mechanical information that had been derived from the current study. Abdominal muscle contraction was simulated in whole cadavers in both the flexed and lateral bending positions to compare the stabilizing effect of the thoracolumbar fascia and intra-abdominal pressure mechanisms. These definitive experiments showed that the resistance to bending in the sagittal plane offered by the abdominal muscles acting through fascial tension was of a similar magnitude to that offered by a raised intra-abdominal pressure, both being relatively small in the fully flexed position. The stabilizing influence of the middle layer of the thoracolumbar fascia in lateral bending was clearly demonstrated and warrants further study in vivo.