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 chemomechanical cycle of this nanomotor during protein translocation. In this study, Förster 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 μM 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 monomerbiased 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 nucleotidebinding 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.Proteins are secreted across or integrated into biological membranes by means of a variety of protein translocation systems that have been characterized over the past several decades. In Escherichia coli, the major pathway for protein secretion is the general secretion (Sec) pathway that is composed of two fundamental components: the SecYEG heterotrimeric complex that comprises the protein-conducting channel and the SecA ATPase nanomotor that drives † This work was supported by Grants GM42033 and GM37639 from Figure S1), binding affinity of unlabeled SP2 and IANBD-labeled SP2 with IAEDANS-labeled SecA-Cys-256 ( Figure S2) and mapping of the FRET-determined signal peptide-binding site on the SecA crystal structures of B. subtilis, E. coli, T. thermophilus, and Mycobacterium tuberculosis ( Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 7. Recently, a model for preprotein translocation has been proposed on the basis of a SecASecYEG cocrystal structure (10) and disulfide cross-linking studies (11). In this model, SecA captures the preprotein in a clamp formed by nucleotide-binding domain 2 (NBD-2) 1 , the preprotein binding domain (PPXD), and the h...

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.

Identification of the signal peptide‐binding site within SecA is critical for understanding the chemo‐mechanical cycle of the SecA nanomotor during protein translocation. While recent studies have addressed this topic, the precise signal‐peptide binding site on SecA remains controversial. We are attempting to identify the SecA signal peptide‐binding site using Fluorescence Resonance Energy Transfer (FRET), which should provide a more global view of the binding site and be less limited than genetic approaches that often eliminate consecutive amino acid sequences and create inactive proteins. This study employs a collection of functional, monocysteine SecA mutants that are labeled with a donor fluorophore (IAEDANS) along with a cysteine‐containing alkaline phosphatase signal peptide (PhoA) that carries the acceptor fluorophore (IANBD). We have optimized the system and determined the equilibrium binding constant of IANBD‐labeled PhoA signal peptide for SecA using fluorescence anisotropy. We are currently working to acquire a sufficient data set to definitively map the signal peptide‐binding site of SecA protein. Future work will address the need to confirm our results utilizing appropriately engineered mutations, which will be tested for alterations in signal peptide binding affinity. Funding provided by National Institute of Health

Self-assembly of β-sheet domains resulting in the formation of pathogenic fibrillar protein aggregates (amyloids) is the causative factor in Alzheimer's, Huntington's, and Creutzfeldt-Jakob diseases. Even though different kinds of protein sequences are known to form toxic structures (amyloids), the underlying mechanism whereby protein aggregation leads to amyloid structure formation is yet to be explained clearly. In this investigation, we have shown that a simple polypeptide, poly(Leu-Glu), on aging in an aqueous solution undergoes structural transition from the soluble monomeric form to amyloid structured aggregates. Water-soluble poly(Leu-Glu) peptide slowly self-assembles into fibrillar structures as evidenced by Fourier transform infrared and circular dichroism spectroscopic methods and thioflavin T binding assay. The polypeptide underwent conformational change from a structure with a mixture of coil & turn to a macromolecular β-sheet conformation and formed amyloid-like fibrils. The fibrils were examined by Congo red staining and transmission electron microscopy. The results indicate that transition from a disordered to an ordered tertiary structure consists of both fundamental helical turns and β-sheet repeats. The comprehensive spectroscopic and microscopic studies suggest that synthetic poly(Leu-Glu) is capable of forming an amyloid-like structure.

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