Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions.

Chromatin is composed of nucleosomes, the universally repeating protein-DNA complex in eukaryotic cells. The crystal structure of the nucleosome core particle from Saccharomyces cerevisiae reveals that the structure and function of this fundamental complex is conserved between single-cell organisms and metazoans. Our results show that yeast nucleosomes are likely to be subtly destabilized as compared with nucleosomes from higher eukaryotes, consistent with the idea that much of the yeast genome remains constitutively open during much of its life cycle. Importantly, minor sequence variations lead to dramatic changes in the way in which nucleosomes pack against each other within the crystal lattice. This has important implications for our understanding of the formation of higher order chromatin structure and its modulation by post-translational modifications. Finally, the yeast nucleosome core particle provides a structural context by which to interpret genetic data obtained from yeast. Coordinates have been deposited with the Protein Data Bank under accession number 1ID3.

Sequence-specific recognition of DNA in the nucleosome by pyrrole-imidazole polyamides.

The ability of DNA-binding proteins to recognize their cognate sites in chromatin is restricted by the structure and dynamics of nucleosomal DNA, and by the translational and rotational positioning of the histone octamer. Here, we use six different pyrrole-imidazole polyamides as sequence-specific molecular probes for DNA accessibility in nucleosomes. We show that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible and that nucleosomes remain fully folded upon ligand binding. Polyamides only failed to bind where sites are completely blocked by interactions with the histone octamer. Removal of the amino-terminal tails of either histone H3 or histone H4 allowed these polyamides to bind. These results demonstrate that much of the DNA in the nucleosome is freely accessible for molecular recognition in the minor groove, and also support a role for the amino-terminal tails of H3 and H4 in modulating accessibility of nucleosomal DNA.

Synthesis, characterization, solution stability, and X-ray crystal structure of the thiolatocobalamin gamma-glutamylcysteinylcobalamin, a dipeptide analogue of glutathionylcobalamin: insights into the enhanced Co-S bond stability of the natural product glutathionylcobalamin.

Glutathionylcobalamin (gamma-glutamylcysteinylglycinylcobalamin; gamma-GluCysGly-Cbl) is a natural product which functions as an intermediate in the biosynthesis of the active B(12) coenzymes adenosylcobalamin and methylcobalamin. Of interest to the present studies is glutathionylcobalamin's unique stability in comparison to other thiolatocobalamins, notably the > or =6 x 10(4) fold less stable cysteinylcobalamin, Cys-Cbl. In order to determine which parts of the glutathione tripeptide contribute to the overall stability of glutathionylcobalamin, two cysteine-containing dipeptides, which are truncated versions of glutathione, were used to synthesize their corresponding cobalamins, specifically gamma-glutamylcysteinylCbl (gamma-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl). As with glutathionylCbl, the dipeptide gamma-GluCys-Cbl forms a stable thiolatocobalamin. However and most interestingly, CysGly-Cbl is observed to be unstable much like Cys-Cbl. The results require that the extra stability of glutathionylcobalamin and its congeners, compared to cysteinylcobalamin and its analogues, must be derived from destabilization by the gamma-NH(3)(+) group in cysteinylcobalamin, or stabilization by the gamma-NHC(=O)- amide linkage in glutathionylcobalamin, or both. To probe any ground-state structural basis for the possible stabilization in gamma-GluCys-containing cobalamins, gamma-GluCys-Cbl was crystallized and yielded the first X-ray structural determination of a true thiolatocobalamin, and only the second structure of a cobalamin containing a Co-S bond, the first example being Randaccio and co-workers' 1999 structure of the thioketone complex, thioureacobalamin, (NH(2))(2)CSCbl. Key features of the structure of gamma-glutamylcysteinylcobalamin include (i) a normal Co-S bond length of 2.267(2) A, (ii) a Co-N(axial) bond length of 2.049(6) A, (iii) two alternate conformations of the gamma-glutamylcysteinyl moiety, and (iv) folding of the corrin ring upward by 24.2 degrees, the highest degree of folding yet observed for a cobalamin. These results do not show any strong stabilization (e.g., no shortened Co-S bond), although it is not clear for certain what the effect is (stabilizing or destabilizing) of the elongated Co-N(axial) bond; instead, the crystallographic results suggest that the metastable Cys-Cbl probably has a Co-S cleavage transition state that is stabilized (along with, possibly, any ground-state destabilization of the Co-S bond). Overall, the results strongly suggest that placing a positive charge on the gamma-NH(3)(+) stabilizes the Co-S bond cleavage transition state, thereby setting the stage for the needed full thermolysis product and kinetic studies-as a function of the axial-base on-off equilibrium-that will be required to understand in even greater detail the unique stability of glutathionyl- (gamma-glutamylcysteinylglycinyl-) and gamma-glutamylcysteinylcobalamins.

Crystal structure of a nucleosome core particle containing the variant histone H2A.Z.

Activation of transcription within chromatin has been correlated with the incorporation of the essential histone variant H2A.Z into nucleosomes. H2A.Z and other histone variants may establish structurally distinct chromosomal domains; however, the molecular mechanism by which they function is largely unknown. Here we report the 2.6 A crystal structure of a nucleosome core particle containing the histone variant H2A.Z. The overall structure is similar to that of the previously reported 2.8 A nucleosome structure containing major histone proteins. However, distinct localized changes result in the subtle destabilization of the interaction between the (H2A.Z-H2B) dimer and the (H3-H4)(2) tetramer. Moreover, H2A.Z nucleosomes have an altered surface that includes a metal ion. This altered surface may lead to changes in higher order structure, and/or could result in the association of specific nuclear proteins with H2A.Z. Finally, incorporation of H2A.Z and H2A within the same nucleosome is unlikely, due to significant changes in the interface between the two H2A.Z-H2B dimers.

Adenosylcobalamin-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii. Rapid, improved purification involving dGTP-based affinity chromatography plus biophysical characterization studies demonstrating enhanced, "crystallographic level" purity.

Ribonucleoside triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii is a 5'-deoxyadenosylcobalamin-dependent (AdoCbl; Coenzyme B12) enzyme. RTPR is also a prototypical adenosylcobalamin-dependent ribonucleotide reductase, one that, as its name indicates, converts ribonucleoside triphosphates (NTP) to deoxyribonucleoside triphosphates (dNTP). Upon substrate binding to RTPR, AdoCbl's cobalt-carbon bond is cleaved to generate cob(II)alamin, 5'-deoxyadenosine, and the cysteine (C408) derived thiyl radical. Five key cysteines (Cys 119, 408, 419, 731, and 736), from among the ten total cysteines, are involved in RTPR's catalytic mechanism. A critical examination of the RTPR isolation and purification literature suggested that the purification protocol currently used results in RTPR which contains 2040% microheterogeneity, along with minor contamination by other proteins. In addition, no report of crystalline RTPR has ever appeared. The literature indicates that irreversible cysteine oxidation (e.g., to -SO2H or -SO3H) is one highly plausible reason for the microheterogeneity of RTPR. The literature also indicates that improvement in the level of enzyme purity is the most effective next step in coaxing enzymes to crystallize that have previously failed to do so. A shortened, improved purification of RTPR has been developed, one involving a shorter purification time, a lower pH, a higher concentration of the more effective reductant DTT (all designed to help protect the cysteines from oxidation), and a final step utilizing our recently reported, improved dGTP-based affinity chromatography resin. The resultant RTPR is approximately 20-30% higher in both specific activity and in its ability to undergo single turnovers, and is homogeneous by mass spectrometry and dynamic light scattering. Additionally, the revised purification procedure eliminates > 30 proteins present in 2-3% amounts along with damaged RTPR that does not bind properly (i.e. tightly) to the dGTP-affinity resin. Finally, dGTP-based affinity chromatography purified RTPR has yielded the first reported, albeit small, single crystals of RTPR.

Synthesis of gamma-phosphate-linked nucleoside affinity chromatography resins for protein purification, including ribonucleoside triphosphate reductase.

Seven nucleotides linked through the gamma-phosphate to diamine hydrocarbons were synthesized and coupled to Sepharose for use in protein purification affinity chromatography. The synthesis involved converting the nucleotides to nucleoside-5'- trimetaphosphates using dicyclohexyl carbodiimide, followed by nucleophilic ring opening of the trimetaphosphate with an alpha, omega-diamino hydrocarbon to generate a gamma-phosphoamide linkage in each nucleotide.

Conversion of D-arabinose to D-erythroascorbic acid and oxalic acid in Sclerotinia sclerotiorum.

D-glycero-Pent-2-enono-1,4-lactone (trivial name: D-erythroascorbic acid) occurs in the phytopathogen, Sclerotinia sclerotiorum (Lib.) de Bary, where it has a potential role as precursor of oxalic acid. On Glc/yeast/malt medium, S. sclerotiorum produces only nominal amounts of D-erythroascorbic acid but even partial replacement of Glc by D-Ara increases production of erythroascorbic acid and oxalic acid. Use of D-[1-14C]-, -[3-14C]-, or -[6-14C]Glc and D-[5-3H]-, -[2-14C,5-3H]-, or -[UL-14C]Ara provide additional information on erythroascorbic acid biosynthesis and cleavage. The latter process resembles that obtained by peroxygenation of erythroascorbic acid in alkaline solution. An unknown erythroascorbic acid-like compound also occurs in both Glc- and Ara-based cultures.