Background: Membrane fusion and fission events are effected by remodeling proteins. Results: Using cryoelectron microscopy, we observed the conversion of large spherical lipid vesicles into narrow protein-coated tubes. Conclusion: Tubulation is accompanied by ␣-synuclein switching into an extended ␣-helical conformation. Significance: The cylindrical micelles produced resemble a hemi-fission/fusion state of the membrane.

BackgroundIn Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays.Methodology/Principal FindingsBy performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein.Conclusions/SignificanceThe temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease.

Background:The mechanism behind diabetes-associated membrane damage by islet amyloid polypeptide (IAPP) is poorly understood. Results: IAPP induces and senses membrane curvature under conditions associated with membrane damage and binds to mitochondrial cristae in vivo. Conclusion: IAPP is a membrane-remodeling and curvature-sensing protein.Significance: Aberrant membrane remodeling could inform disruption of membrane integrity in diabetes and perhaps other amyloid diseases.

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Ameloblastin (Ambn), the most abundant non-amelogenin enamel protein, is intrinsically disordered and has the potential to interact with other enamel proteins and with cell membranes. Here, through multiple biophysical methods, we investigated the interactions between Ambn and large unilamellar vesicles (LUVs), whose lipid compositions mimicked cell membranes involved in epithelial cell-extracellular matrix adhesion. Using a series of Ambn Trp/Phe variants and Ambn mutants, we further showed that Ambn binds to LUVs through a highly conserved motif within the sequence encoded by exon 5. Synthetic peptides derived from different regions of Ambn confirmed that the sequence encoded by exon 5 is involved in LUV binding. Sequence analysis of Ambn across different species showed that the N-terminus of this sequence contains a highly conserved motif with a propensity to form an amphipathic helix. Mutations in the helix-forming sequence resulted in a loss of peptide binding to LUVs. Our in vitro data suggest that Ambn binds the lipid membrane directly through a conserved helical motif and have implications for biological events such as Ambn-cell interactions, Ambn signaling, and Ambn secretion via secretory vesicles.