Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cisorientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or diseaseassociated changes in copper homeostasis likely go along with altered APP synaptic function.

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken alpha-spectrin SH3 domain (62 residues), alphaB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Calpha, Cbeta, C' and N resonances in the core domain of alphaB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).

The asymmetric distribution of lipids between the two bilayer leaflets represents a typical feature of biological membranes. The loss of this asymmetry, for example the exposure of negatively charged lipids on the extracellular membrane leaflet of mammalian cells, is involved in apoptosis and occurs in tumor cells. Thus, the controlled production of asymmetric liposomes helps to better understand such crucial cellular processes. Here, we present an approach that allows us to design asymmetric model-membrane experiments on a rational basis and predict the fraction of exchanged lipid. In addition, we developed a label-free and nondestructive assay to quantify the asymmetric uptake of negatively charged lipids in terms of the zeta potential. This significantly enhances the applicability, impact, and predictive power of model membranes.

Separation of cells and organelles by bilayer membranes is a fundamental principle of life. Cellular membranes contain a baffling variety of proteins, which fulfil vital functions as receptors and signal transducers, channels and transporters, motors and anchors. The vast majority of membrane-bound proteins contain bundles of alpha-helical transmembrane domains. Understanding how these proteins adopt their native, biologically active structures in the complex milieu of a membrane is therefore a major challenge in today's life sciences. Here, we review recent progress in the folding, unfolding and refolding of alpha-helical membrane proteins and compare the molecular interactions that stabilise proteins in lipid bilayers. We also provide a critical discussion of a detergent denaturation assay that is increasingly used to determine membrane-protein stability but is not devoid of conceptual difficulties.

Typical Kondo insulators (KIs) can have a nontrivial Z_{2} topology because the energy gap opens at the Fermi energy (E_{F}) by a hybridization between odd- and even-parity bands. SmB_{6} deviates from such KI behavior, and it has been unclear how the insulating phase occurs. Here, we demonstrate that charge fluctuations are the origin of the topological insulating phase in SmB_{6}. Our angle-resolved photoemission spectroscopy results reveal that with decreasing temperature the bottom of the d-f hybridized band at the X[over ¯] point, which is predicted to have odd parity and is required for a topological phase, gradually shifts from below to above E_{F}. We conclude that SmB_{6} is a charge-fluctuating topological insulator.