The dengue virus NS2B-NS3 protease retains the closed conformation in the complex with BPTI.

The C-terminal β-hairpin of NS2B (NS2Bc) in the dengue virus NS2B-NS3 protease is required for full enzymatic activity. In crystal structures without inhibitor and in the complex with bovine pancreatic trypsin inhibitor (BPTI), NS2Bc is displaced from the active site. In contrast, nuclear magnetic resonance (NMR) studies in solution only ever showed NS2Bc in the enzymatically active closed conformation.

Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, epsilon, θ and β reveals a highly flexible arrangement of the proofreading domain.

A complex of the three (αεθ) core subunits and the β2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site.

Biosynthetically directed ²H labelling for stereospecific resonance assignments of glycine methylene groups.

Stereospecific resonance assignments of the α-protons of glycine are often difficult to obtain by measurements of scalar coupling constants or nuclear Overhauser effects. Here we show that these stereospecific resonance assignments can readily be obtained by cell-free protein synthesis in D(2)O, as the serine hydroxymethyltransferase, that is naturally present in E. coli cell extracts, selectively replaces the pro-2S proton of glycine by a deuterium.

Tunable paramagnetic relaxation enhancements by [Gd(DPA)(3)] (3-) for protein structure analysis.

Paramagnetic relaxation enhancements (PRE) present a powerful source of structural information in nuclear magnetic resonance (NMR) studies of proteins and protein-ligand complexes. In contrast to conventional PRE reagents that are covalently attached to the protein, the complex between gadolinium and three dipicolinic acid (DPA) molecules, [Gd(DPA)(3)](3-), can bind to proteins in a non-covalent yet site-specific manner. This offers straightforward access to PREs that can be scaled by using different ratios of [Gd(DPA)(3)](3-) to protein, allowing quantitative distance measurements for nuclear spins within about 15 A of the Gd(3+) ion.

[Ln(DPA)(3)](3-) is a convenient paramagnetic shift reagent for protein NMR studies.

Paramagnetic lanthanide ions present outstanding tools for structural biology by NMR spectroscopy. Here we show that the 3:1 complexes between dipicolinic acid and lanthanides are paramagnetic reagents which can site-specifically bind to a wide range of proteins without formation of a covalent bond. The observed pseudocontact shifts can be interpreted by a single magnetic susceptibility anisotropy tensor, enabling its use for structure refinements.

A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader.

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication.

Single-molecule studies of fork dynamics in Escherichia coli DNA replication.

We present single-molecule studies of the Escherichia coli replication machinery. We visualize individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis.

Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primase.

DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB.

Crystal and solution structures of the helicase-binding domain of Escherichia coli primase.

During bacterial DNA replication, the DnaG primase interacts with the hexameric DnaB helicase to synthesize RNA primers for extension by DNA polymerase. In Escherichia coli, this occurs by transient interaction of primase with the helicase. Here we demonstrate directly by surface plasmon resonance that the C-terminal domain of primase is responsible for interaction with DnaB6.