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Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, [...]

A study was conducted to characterize the crystal structure of ErmC', an rRNA methyltransferase from Bacillus subtilis which mediates antibiotic resistance in bacteria. Polymerase chain reaction amplification was carried out on the ermC' gene and the upstream kdsB cistron to determine the expression vector pTERM31. The ErmC' supported maximum flexibility at the amino terminus and in the loop between beta-6 and beta-7.

The folding kinetics of FK506 binding protein (FKBP), a prolyl isomerase, are studied. The primary finding is that FKBP catalyzes its own folding. The FKBP refolding kinetics are composed of three exponential phases; a fast phase, an intermediate phase and a slow phase. Unfolding happens in the intermediate phase. Findings show that the slow phase in FKBP folding is an autocatalyzed formation of native FKBP from kinetically trapped species with non-native proline isomers.

The structure of the DNA-binding domain of human papillomavirus E2 protein in free solution comprises two pairs of alpha-helices and an eight-stranded beta-barrel. Heteronuclear multidimensional NMR spectroscopic analysis of the crystal structure of the protein-DNA complex reveals disorders in a beta-hairpin attached to DNA. Steady-state 1H-15N heteronuclear Overhauser effect measurements reveal that the disordered region is active in the absence of DNA. The human papillomavirus E2 protein shares a 31% amino acid similarity with the bovine virus E2 protein.

ErmC' methyltransferase activity in Bacillus subtilis requires a 262-nucleotide RNA fragment from B. subtilis 23S rRNA's domain V for the methylation substrate. Both Escherichia coli and B. subtilis use the methyltransferase to become resistant to the antibiotic macrolide-lincosamide-streptogramin B group. ErmC' expression was generated using translational coupling in a dicistronic vector with kdsB as the upstream gene.

Heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy and a distance geometry/stimulated annealing protocol were used to determine the 3-dimensional solution structure of FK-binding protein (FKBP)/ascomycin complex. The FKBP/ascomycin complex is implicated in the immunosuppressive abilities of FK506 against FKBP. The results of comparisons of the FKBP/ascomycin complex structure with NMR-derived solution structures of unbound FKBP and the X-ray crystal structure FKBP/FK506 complex are also presented.

Heteronuclear three-dimensional nuclear magnetic resonance (NMR) spectroscopy was used to determine the solution structure of the cyclosporin A/cyclophilin complex. The structure of the complex differs considerably from previous models. Knowledge of this structure adds to that for the uncomplexed molecules and may shed light on the function of cyclosporin A as an immunosuppressant.

Several NMR studies are conducted to present the structural characterization of the soluble amyloid [beta]-peptide oligomers. These oligomers are shown to be highly stable and even have the capability of altering the synaptic activity.

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultra-centrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 °C, and a minor transition at 58 °C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at ∼75 kDa and the other at ∼55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 °C transition, and a loose structure of the C-terminus, giving rise to the 39 °C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid beta-peptides that can form insoluble fibrils or soluble oligomeric assemblies. Soluble forms are present in the brains and tissues of Alzheimer's patients, and their presence correlates with disease progression. Long-lived soluble forms can be generated in vitro by using small amounts of aliphatic hydrocarbon chains of detergents or fatty acids in preparations of amyloid beta-peptides. Using NMR, we have characterized soluble oligomers of Abeta preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel beta-sheet structure that is different from fibrils which contain only parallel beta-sheets. Using the structural data, we engineered a disulfide bond into the soluble Abeta globulomer to give a "new" soluble antigen that is stable, homogeneous, and binds with the same affinity to selective antibodies as the parent wt globulomer.