Analysis of monomeric Abeta (1-40) peptide by capillary electrophoresis.

A method was developed to characterize and quantify preparations of monomeric beta-amyloid (Abeta) peptide using capillary electrophoresis (CE) with UV absorbance detection. The detection limit for Abeta monomer using this method was 0.5 microM (19 pg). The self-assembly of Abeta to form amyloid fibrils is closely linked to Alzheimer's disease and is the subject of intense investigations. Consistent preparation of Abeta monomer samples at known concentrations and free of aggregates is a significant challenge for researchers studying the mechanism of Abeta fibril formation and searching for small molecules that inhibit Abeta fibril formation. Samples of Abeta monomer are known to sometimes contain pre-existing aggregates that can affect the kinetics and structure of amyloid fibrils. The CE method presented here showed that some of the monomeric Abeta samples prepared for this study contained a species producing a second peak (in addition to the major monomer peak). The aggregation was monitored using a thioflavin T fluorescence assay, and the resulting fibrils were characterized by transmission electron microscopy. Monomer samples containing the additional peak based on CE analysis were shown to aggregate more rapidly than monomer samples that were free of this putative Abeta aggregate peak.

Mapping of the signal peptide-binding domain of Escherichia coli SecA using Förster resonance energy transfer.

Identification of the signal peptide-binding domain within SecA ATPase is an important goal for understanding the molecular basis of SecA preprotein recognition as well as elucidating the chemo-mechanical cycle of this nanomotor during protein translocation. In this study, Forster resonance energy transfer methodology was employed to map the location of the SecA signal peptide-binding domain using a collection of functional monocysteine SecA mutants and alkaline phosphatase signal peptides labeled with appropriate donor-acceptor fluorophores. Fluorescence anisotropy measurements yielded an equilibrium binding constant of 1.4 or 10.7 muM for the alkaline phosphatase signal peptide labeled at residue 22 or 2, respectively, with SecA, and a binding stoichiometry of one signal peptide bound per SecA monomer. Binding affinity measurements performed with a monomer-biased mutant indicate that the signal peptide binds equally well to SecA monomer or dimer. Distance measurements determined for 13 SecA mutants show that the SecA signal peptide-binding domain encompasses a portion of the preprotein cross-linking domain but also includes regions of nucleotide-binding domain 1 and particularly the helical scaffold domain. The identified region lies at a multidomain interface within the heart of SecA, surrounded by and potentially responsive to domains important for binding nucleotide, mature portions of the preprotein, and the SecYEG channel. Our FRET-mapped binding domain, in contrast to the domain identified by NMR spectroscopy, includes the two-helix finger that has been shown to interact with the preprotein during translocation and lies at the entrance to the protein-conducting channel in the recently determined SecA-SecYEG structure.