No abstract

An interhelical distance has been precisely measured by REDOR solid-state NMR spectroscopy in the transmembrane tetrameric bundle of M2-TMP, from the M2 proton channel of the influenza A viral coat. The high-resolution structure of the helical backbone has been determined using orientational restraints from uniformly aligned peptide preparations in hydrated dimyristoylphosphatidylcholine bilayers. Here, the distance between (15)N(pi) labeled His37 and (13)C(gamma) labeled Trp41 is determined to be less than 3.9 A. Such a short distance, in combination with the known tilt and rotational orientation of the individual helices, permits not only a determination of which specific side chain pairings give rise to the interaction, but also the side chain torsion angles and restraints for the tetrameric bundle can also be characterized. The resulting proton channel structure is validated in a variety of ways. Both histidine and tryptophan side chains are oriented in toward the pore where they can play a significant functional role. The channel appears to be closed by the proximity of the four indoles consistent with electrophysiology and mutagenesis studies of the intact protein at pH 7.0 and above. The pore maintains its integrity to the N terminal side of the membrane, and at the same time, a cavity is generated that appears adequate for binding amantadine. Finally, the observation of a 2 kHz coupling in the PISEMA spectrum of (15)N(pi)His37 validates the orientation of the His37 side chain based on the observed REDOR distance.

Dendritic cell-based cancer immunotherapy requires tumour antigens to be delivered efficiently into dendritic cells and their migration to be monitored in vivo. Nanoparticles have been explored as carriers for antigen delivery, but applications have been limited by the toxicity of the solvents used to make nanoparticles, and by the need to use transfection agents to deliver nanoparticles into cells. Here we show that an iron oxide-zinc oxide core-shell nanoparticle can deliver carcinoembryonic antigen into dendritic cells while simultaneously acting as an imaging agent. The nanoparticle-antigen complex is efficiently taken up by dendritic cells within one hour and can be detected in vitro by confocal microscopy and in vivo by magnetic resonance imaging. Mice immunized with dendritic cells containing the nanoparticle-antigen complex showed enhanced tumour antigen specific T-cell responses, delayed tumour growth and better survival than controls.

We have observed a common sequence motif in membrane proteins, which we call a glycine zipper. Glycine zipper motifs are strongly overrepresented and conserved in membrane protein sequences, and mutations in glycine zipper motifs are deleterious to function in many cases. The glycine zipper has a significant structural impact, engendering a strong driving force for right-handed packing against a neighboring helix. Thus, the presence of a glycine zipper motif leads directly to testable structural hypotheses, particularly for a subclass of glycine zipper proteins that form channels. For example, we suggest that the membrane pores formed by the amyloid-␤ peptide in vitro are constructed by glycine zipper packing and find that mutations in the glycine zipper motif block channel formation. Our findings highlight an important structural motif in a wide variety of normal and pathological processes.amyloid-␤ ͉ membrane channel ͉ membrane protein structure ͉ prion ͉ transmembrane helix M ore than 13 structures a day are currently being deposited in the Protein Data Bank (1), and structural genomics centers have been created to obtain structures even faster [such as the National Institute of General Medical Sciences (NIGMS) Protein Structure Initiative, www.nigms.nih.gov͞psi]. In this assault on protein structure, however, technical challenges have left membrane proteins far behind. Membrane protein structures are currently being solved at Ϸ0.2% of the pace of soluble proteins (2). Thus, membrane protein biochemists are relatively starved for structural insight into these key proteins. In the absence of dramatic technical improvements, alternatives to experimental structure determination are needed.Here, we describe a transmembrane (TM) sequence motif, the glycine zipper, that can lead directly to structural models for many membrane proteins. The most significant glycine zipper sequence patterns are (G,A,S)XXXGXXXG and GXXXGX-XX(G,S,T). These patterns contain a GXXXG motif, which is known to be important in TM helix homodimers where the Gly faces are in direct contact (3-5). The GXXXG sequence pattern is statistically overrepresented in membrane proteins in general, not just in TM homodimers (4). Nevertheless, the structural role of the GXXXG pattern in other types of TM helix packing interactions has not been elucidated. We find that the addition of an appropriately spaced small residue, as found in the glycine zipper, leads to a distinct preference for right-handed packing against a heterologous helix surface. Thus, the presence of a glycine zipper generates a strong helix packing prediction, particularly for homooligomeric channel proteins, providing a structural foundation for hypothesis-driven investigations. MethodsGlycine Zipper Motif Search. We started with Swiss-Prot release 41.15 containing 129,996 proteins (6). All sequences Ͻ50 residues in length were removed, leaving 125,887 proteins. Helical membrane proteins were identified by using the Eisenberg hydrophobicity scale and a window length of 21 residues (7). ...