Thermal and sodium dodecylsulfate induced transitions of streptavidin

The strong specific binding of streptavidin (SA) to biotin is utilized in numerous biotechnological applications. The SA tetramer is also known to exhibit significant stability, even in the presence of sodium dodecylsulfate (SDS). Despite its importance, relatively little is known about the nature of the thermal denaturation pathway for SA. This work uses a homogeneous SA preparation to expand on the data of previous literature reports, leading to the proposal of a model for temperature induced structural changes in SA. Temperature dependent data were obtained by SDS and native polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and fluorescence and ultraviolet (UV)-visible spectroscopy in the presence and absence of SDS. In addition to the development of this model, it is found that the major thermal transition of SA in 1% SDS is reversible. Finally, although SA exhibits significant precipitation at elevated temperatures in aqueous solution, inclusion of SDS acts to prevent SA aggregation.     

A novel non-radioactive primase–pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG

Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase–pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z′ = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery.     

Small G—protein RhoA is a potential inhibitor of cardiac fast sodium current

Journal of Physiology and Biochemistry - Small G-proteins of Rho family modulate the activity of several classes of ion channels, including K+ channels Kv1.2, Kir2.1, and ERG; Ca2+ channels; and...     

The C-terminal domain of human Rev1 contains independent binding sites for DNA polymerase η and Rev7 subunit of polymerase ζ

Human Rev1 is a translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap-filling. Rev1 plays an essential structural role in TLS by providing a binding platform for other TLS polymerases that insert nucleotides across DNA lesions (polη, polι, polκ) and extend the distorted primer-terminus (pol?). We use NMR spectroscopy to demonstrate that the Rev1 C-terminal domain utilizes independent interaction interfaces to simultaneously bind a fragment of the ’inserter’ polη and Rev7 subunit of the ’extender’ pol?, thereby serving as a cassette that may accommodate several polymerases making them instantaneously available for TLS.

Structured summary of protein interactions

REV1, REV3 and REV7physically interact by nuclear magnetic resonance (View interaction), molecular sieving (View interaction) and isothermal titration calorimetry (View interaction).REV3 and REV7bind by molecular sieving (View interaction)REV1 and Polη-RIR peptidebind by nuclear magnetic resonance (View interaction)REV1, REV3, REV7 and Polη-RIR peptidephysically interact by nuclear magnetic resonance (View interaction).     

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.     

Effects of Exogenously Applied Gibberellins and Thidiazuron on Phytohormone Profiles of Long-Shoot Buds and Cone Gender Determination in Lodgepole Pine

In long-shoot buds of lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm), cone-bud initiation and gender differentiation occur in a site-specific manner: female cone buds are normally restricted to the distal portion, whereas male cone buds are located in the proximal portion. Application of a paste containing two plant growth regulators gibberellins A₄ + A₇ (GA_4/7) combined with thidiazuron (TDZ) to long-shoot buds prior to cone-bud gender determination altered endogenous phytohormone profiles and induced female cone-bud formation in the proximal portion of the long-shoot bud, where male cone buds normally occur. Induced cone clusters observed in the following spring were either entirely female or a mixture of both female and male cones. Endogenous phytohormones in the long-shoot bud tissues were quantified by the stable isotope dilution method using high performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction monitoring mode. Applied GA_4/7 + TDZ led to increased concentrations of endogenous zeatin-type cytokinins, that is, trans-zeatin riboside and dihydrozeatin riboside, whereas concentrations of abscisic acid (ABA) and its catabolite, ABA glucose ester, were decreased, all relative to control, in untreated long-shoot bud tissue. Concentrations of extractable GA₄ and GA₇ declined in long-shoot bud tissues over 4 weeks following treatment with exogenous GA_4/7. This study demonstrates that high levels of endogenous zeatin-type cytokinins, together with reduced levels of ABA, both induced by applied GA_4/7 + TDZ, are positively associated with an increased female cone-bud formation in long-shoot buds.