Routine single particle CryoEM sample and grid characterization by tomography.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

Characterization of flavonol inhibition of DnaB helicase: real-time monitoring, structural modeling, and proposed mechanism.

DnaB helicases are motor proteins essential for DNA replication, repair, and recombination and may be a promising target for developing new drugs for antibiotic-resistant bacteria. Previously, we established that flavonols significantly decreased the binding ability of Klebsiella pneumoniae DnaB helicase (KpDnaB) to dNTP. Here, we further investigated the effect of flavonols on the inhibition of the ssDNA binding, ATPase activity, and dsDNA-unwinding activity of KpDnaB. The ssDNA-stimulated ATPase activity of KpDnaB was decreased to 59%, 75%, 65%, and 57%, in the presence of myricetin, quercetin, kaempferol, and galangin, respectively. The ssDNA-binding activity of KpDnaB was only slightly decreased by flavonols. We used a continuous fluorescence assay, based on fluorescence resonance energy transfer (FRET), for real-time monitoring of KpDnaB helicase activity in the absence and presence of flavonols. Using this assay, the flavonol-mediated inhibition of the dsDNA-unwinding activity of KpDnaB was observed. Modeled structures of bound and unbound DNA showed flavonols binding to KpDnaB with distinct poses. In addition, these structural models indicated that L214 is a key residue in binding any flavonol. On the basis of these results, we proposed mechanisms for flavonol inhibition of DNA helicase. The resulting information may be useful in designing compounds that target K. pneumoniae and other bacterial DnaB helicases.

DNA binding activity of Helicobacter pylori DnaB helicase: the role of the N-terminal domain in modulating DNA binding activities.

Replicative helicases are major motor proteins essential for chromosomal DNA replication in prokaryotes. Usually hexameric in solution, their DNA binding property must have different roles at stages ranging from the loading onto a branched structure at initiation from the origin to the highly processive translocation during elongation. Here, we have analysed the DNA binding activity of Helicobacter pylori (Hp) replicative helicase, DnaB. The results indicate that while the C-terminal region is important for its DNA binding activity, the N-terminus appears to dampen the protein's affinity for DNA. The masking activity of the N-terminus does not require ATP or hexamerization of HpDnaB and can be overcome by deleting the N-terminus. It can also be neutralized by engaging the N-terminus in an interaction with a partner like the C-terminus of DnaG primase. The inhibitory effect of the N-terminus on DNA binding activity is consistent with the 3D homology model of HpDnaB. Electron microscopy of the HpDnaB-ssDNA complex showed that HpDnaB preferentially bound at the ends of linear ssDNA and translocated along the DNA in the presence of ATP. These results provide an insight into the stimulatory and inhibitory effects of different regions of HpDnaB on DNA binding activities that may be central to the loading and translocation functions of DnaB helicases.

Effect of protein stabilization on charge state distribution in positive- and negative-ion electrospray ionization mass spectra.

Changes in protein conformation are thought to alter charge state distributions observed in electrospray ionization mass spectra (ESI-MS) of proteins. In most cases, this has been demonstrated by unfolding proteins through acidification of the solution. This methodology changes the properties of the solvent so that changes in the ESI-MS charge envelopes from conformational changes are difficult to separate from the effects of changing solvent on the ionization process. A novel strategy is presented enabling comparison of ESI mass spectra of a folded and partially unfolded protein of the same amino acid sequence subjected to the same experimental protocols and conditions. The N-terminal domain of the Escherichia coli DnaB protein was cyclized by in vivo formation of an amide bond between its N- and C-termini. The properties of this stabilized protein were compared with its linear counterpart. When the linear form was unfolded by decreasing pH, a charge envelope at lower m/z appeared consistent with the presence of a population of unfolded protein. This was observed in both positive-ion and negative-ion ESI mass spectra. Under the same conditions, this low m/z envelope was not present in the ESI mass spectrum of the stable cyclized form. The effects of changing the desolvation temperature in the ionization source of the Q-TOF mass spectrometer were also investigated. Increasing the desolvation temperature had little effect on positive-ion ESI mass spectra, but in negative-ion spectra, a charge envelope at lower m/z appeared, consistent with an increase in the abundance of unfolded protein molecules.

Loading a ring: structure of the Bacillus subtilis DnaB protein, a co-loader of the replicative helicase.

Loading of the ring-shaped replicative helicase is a critical step in the initiation of DNA replication. Bacillus subtilis has adopted a two-protein strategy to load its hexameric replicative helicase: DnaB and DnaI interact with the helicase and mediate its delivery onto DNA. We present here the 3D electron microscopy structure of the DnaB protein, along with a detailed analysis of both its oligomeric state and its domain organization. DnaB is organized as an asymmetric tetramer that is comprised of two stacked components, one arranged as a closed collar and the other as an open sigma shape. Intriguingly, the 3D map of DnaB exhibits an overall architecture similar to the structure of the Escherichia coli gamma-complex, the loader of the ring-shaped processivity factor. We propose a model whereby each DnaB monomer participates in both stacked components of the tetramer and displays a different overall shape. This asymmetric quaternary organization could be a general feature of ring loaders.