Three-dimensional structure of acylphosphatase. Refinement and structure analysis.

We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.     

Bacteriophage SPO1 structure and morphogenesis. I. Tail structure and length regulation.

Bacteriophage SPO1, a structually complex phage with hydroxymethyl uracil replacing thymine, has been studied by structural and chemical methods with the aim of defining the virion organization. The contractile tail of SPO1 consists of a complex baseplate, a tail tube, and a 140-nm-long sheath composed of stacked disks (4.1 nm repeat), each containing six subunits of molecular weight 60,300. The subunits are arranged in six parallel helices, each with a helical screw angle (omega 0) of 22.5 degrees. The baseplate was shown to undergo a structural rearrangement during tail contraction into a hexameric pinwheel. A mutation in gene 8 which produced unattached heads and tails also produced tails of different lengths. The tail length distribution suggests that the smallest integral length increment is a single disk of subunits. The structural arrangement of subunits in long tails is identical to that of normal tails, and the tails can contract. Many of the long tails showed partial stain penetration within the tail tube to a point which coincides with the top of a unit-length tail. The implications of these findings with respect to tail length regulation are discussed.     

Bacteriophage SPO1 structure and morphogenesis. II. Head structure and DNA size.

The capsid of bacteriophage SPO1 is icosahedral, and the subunit arrangement on the 87-nm-diameter head suggests the triangulation number T = 16. The major capsid protein (45,700 daltons) is cleaved from a 47,700-dalton precursor. Tubular heads (polyheads) are produced by mutations in genes 5 and 8 and contain cores as well as capped ends. The lattice constant of these structures is 13.4 nm; diameter is 109.5 nm. The size of the double-stranded SPO1 DNA (containing 5' hydroxymethyl uracil in place of thymine) was measured by sedimentation analysis and electron microscopy and has a molecular weight of 86 X 10(6) (about 140 kilobase pairs), which is smaller than several previously reported values.     

Tubulin structure and biochemistry.

In the past year, much has been learned about structure-function correlations in the tubulin molecule, and specifically about the nature and roles of post-translational modifications and tubulin isotypes. The interactions between tubulin and its ligands--both microtubule-associated proteins and anti-mitotic drugs--are becoming clearer at the molecular level.     

Water structure and hydration.

Possible structures adopted by bulk water are discussed with special reference to the possible presence of monomeric water and the detection of 'free' -OH groups. The way in which water tends to accommodate small hydrophobic molecules is considered, with particular reference to the clathrate theory and the phenomenon of 'structure making'. Cage-pairing and cage-sharing processes are described. Consideration of the way water solvates cations and anions is followed by a discussion of the way these solvated ions interact with the bulk medium. Large symmetrical alkylammonium ions probably encourage clathrate cage formation, at least at low temperatures. Particular reference is made to the use of infrared, Raman, ultraviolet, n.m.r. and e.s.r. spectroscopic techniques to the study of water and aqueous solutions.     

Chromatin and nucleosome structure.

Chromatin nucleosomes (mononucleosomes through pentanucleosomes) have been isolated by staphylococcal nuclease digestion of calf thymus nuclei. The peak value ellipticity is the same for all oligomers, 1900 deg cm2, mol-1 at 280-nm, 23 degrees C. The dh280/dT vs T show a progressive increase in Tm of the main thermal band (73.5 degrees C, monomer; 79 degrees C, pentamer). Very small amounts of free DNA can be observed in the melting profiles, and shoulders at 60 degrees C and 93 degrees C appear and increase in magnitude as the particle size increases. The magnitude of the change, delta[theta]280, increases with oligomer size. This pattern could result from an initial unfolding of an asymmetric assembly of nucleosomes (polynucleosome superhelix) in addition to the denaturation of the internal nucleosome structure, and a subsequent or simultaneous denaturation of the double strand DNA. The extent of this unfolding appears to depend upon the size of the oligomer and therefore implies interactions between asymmetrically assembled neighboring nucleosomes.     

Centromere and kinetochore structure.

Recent studies have begun to yield some insight into the structural and regulatory components of centromeres, and new assays have been developed that promise to be of use in advancing our understanding of centromere structure and function. In the budding yeast Saccharomyces cerevisiae new proteins that are required for centromere function have been identified and an in vitro microtubule-binding assay that should assist in dissecting the process of centromere microtubule attachment has been developed. The centromere-specific DNA sequences in the fission yeast Schizosaccharomyces pombe have been identified and partially characterized. In addition, several mammalian centromere proteins have been further characterized, and localization and inhibition studies suggest roles for these proteins in the regulation and assembly of a functional kinetochore.     

Human interstitial retinoid-binding protein. Gene structure and primary structure

Interstitial retinoid-binding protein (IRBP) is synthesized and secreted by rod photoreceptor cells into the interphotoreceptor matrix and is known to bind retinoids and fatty acids. We have used cDNA clones encoding human IRBP to isolate a 15-kilobase genomic fragment that encompasses the complete human IRBP gene. The IRBP gene spans more than 11 kilobases and is interrupted by three introns, all of which are positioned near the 3'-end of the coding sequence. The 3741-base pair coding region of IRBP appears to have been generated by quadruplication of an approximately 900 base pair long ancestral gene. The deduced amino acid sequence predicts a mature protein of 1,230 residues (calculated molecular weight 133,000). The protein sequence can be aligned into four homologous segments, each consisting of about 300 residues. Sequence similarity between segments is as high as 60% when conservative substitutions are taken into account. Two putative N-linked glycosylation sites are located in highly conserved domains in the center of the first and second segment of IRBP. A domain consisting of 41 residues at the COOH-terminal end of the third segment has 15 matching residues (38%) with an intradiscal loop of rhodopsin, a retinal-binding protein in rod photoreceptors.     

Biologic significance of the structure of hydrocarbons. I. Chain structure

1. The biologic experiments with the links of the methane series—n-pentane, n-hexane, n-heptane, n-octane, i-octane, and pentene—gave these qualitative results: (a) The higher the number of CH₂ groups, the longer the chain, the longer the average lifetime of the animal. (b) The ramified chain does not appear to act differently from the saturated straight chain with the same number of C atoms. (c) One double bond within the chain shortens the lifetime to a considerable degree. 2. The quantitative discussion shows that the lifetimes depend exponentially on the molecular weight. 3. Qualitatively the hypothesis is supported that with rising molecular weight the concentration of CH₂ groups within the animal diminishes according to the vapor pressure or the thermodynamic potential. However, lifetime and these physical properties obey different functions. 4. These physical properties are of high biologic importance. But they are not sufficient to explain the biologic effects quantitatively.     

Nucleosome structure.

Electron microscopic and biochemical results are presented supporting the following conclusions: (1) Two molecules of each histone H2A, H2B, H3 and H4 are necessary and sufficient to form a nucleosome with a diameter of 12.5 +/- 1 nm and containing about 200 base pairs of DNA. (2) H3 plus H4 alone can compact 129 +/- 8 DNA base pairs into a sub-nucleosomal particle with a diameter of 8 +/- 1 nm. In such a particle the DNA duplex is under a constraint equivalent to negative superhelicity. (3) Chromatin should be viewed as a dynamic structure, oscillating between a compact structure (the nucleosome) and more open structures, depending on the environmental conditions.     

Expression and structure of cartilage proteins.

Cartilage has unique physical characteristics attributable to the presence of an unusually high content of proteoglycan embedded in the network of collagen fibrils. Advances in understanding the structure of these components and how their synthesis is regulated have been greatly assisted by the application of molecular biology. For example, an immortalized rat chondrocyte cell line was obtained by infection with a recombinant retrovirus encoding the myc gene product. Several positive and negative DNA regulatory elements of the collagen II gene have been identified that appear to be important in the regulation of this gene in chondrocytes. The complete primary structure of the cartilage proteoglycan (aggrecan) core protein deduced from cDNA sequence displays a complex multidomain structure including numerous repeats of Ser-Gly sequences and sequence homologies with link protein and animal lectins. Such studies advance our understanding of normal morphogenetic events and lay the groundwork for determining the basis of molecular and genetic defects.