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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.

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.

It is advocated that kinetic and thermodynamic profiling of bioactive compounds should be incorporated and utilized as complementary tools for hit and lead optimizations in drug discovery. To assess their applications in the EED hit-to-lead optimization process, large amount of thermodynamic and kinetic data were collected and analyzed via isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR), respectively. Slower dissociation rates (koff) of the lead compounds were observed as the program progressed. Analysis of the kinetic data indicated that compound cellular activity correlated with both Ki and koff. Our analysis revealed that ITC data should be interpreted in the context of chiral purity of the compounds. The thermodynamic signatures of the EED aminopyrrolidine compounds were found to be mainly enthalpy driven with improved enthalpic contributions as the program progressed. Our study also demonstrated that significant challenges still exist in utilizing kinetic and thermodynamic parameters for hit selection. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid β-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 β-peptides. Using NMR, we have characterized soluble oligomers of Aβ preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel Aβ-sheet structure that is different from fibrils which contain only parallel Aβ-sheets. Using the structural data, we engineered a disulfide bond into the soluble Aβ 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. [ABSTRACT FROM AUTHOR]

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.

A series of (5-substituted pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidine (C5-Pro-Pro) analogues was discovered as dipeptidyl peptidase IV (DPPIV) inhibitors as a potential treatment of diabetes and obesity. X-ray crystallography data show that these inhibitors bind to the catalytic site of DPPIV with the cyano group forming a covalent bond with the serine residue of DPPIV. The C5-substituents make various interactions with the enzyme and affect potency, chemical stability, selectivity, and PK properties of the inhibitors. Optimized analogues are extremely potent with subnanomolar K(i)'s, are chemically stable, show very little potency decrease in the presence of plasma, and exhibit more than 1,000-fold selectivity against related peptidases. The best compounds also possess good PK and are efficacious in lowering blood glucose in an oral glucose tolerance test in ZDF rats.