The three-dimensional structure of the dimeric transmembrane domain of glycophorin A (GpA) was determined by solution nuclear magnetic resonance spectroscopy of a 40-residue peptide solubilized in aqueous detergent micelles. The GpA membrane-spanning alpha helices cross at an angle of -40 degrees and form a small but well-packed interface that lacks intermonomer hydrogen bonds. The structure provides an explanation for the previously characterized sequence dependence of GpA dimerization and demonstrates that van der Waals interactions alone can mediate stable and specific associations between transmembrane helices.

Elevated reactive oxygen species (ROS) induce the formation of lipids in neurons that are transferred to glia where they form lipid droplets (LD). We show that glial and neuronal monocarboxylate transporters (MCTs), fatty acid transport proteins (FATP), and apolipoproteins are critical for glial LD formation. MCTs enable glia to secrete and neurons to absorb lactate, which is converted to pyruvate and acetyl-CoA in neurons. Lactate metabolites provide a substrate for synthesis of fatty acids, which are processed and transferred to glia by FATP and apolipoproteins. In the presence of high ROS, inhibiting lactate transfer or lowering FATP or apolipoprotein levels all decrease glial LD accumulation in flies and in primary mouse glial-neuronal cultures. We show that human APOE can substitute for a fly glial apolipoprotein and that APOE4, an Alzheimer’s Disease susceptibility allele, is impaired in lipid transport and promotes neurodegeneration, providing insights into disease mechanisms.

Phospholamban is a 52 amino acid calcium regulatory protein found as pentamers in cardiac SR membranes. The pentamers form through interactions between its transmembrane domains, and are stable in SDS. We have employed a saturation mutagenesis approach to study the detailed interactions between the transmembrane segments, using a chimeric protein construct in which staphylococcal nuclease (a monomeric soluble protein) is fused to the N‐terminus of phospholamban. The chimera forms pentamers observable in SDS‐PAGE, allowing the effects of mutations upon the oligomeric association to be determined by electrophoresis. The disruptive effects of amino acid substitutions in the transmembrane domain were classified as sensitive, moderately sensitive or insensitive. Residues of the same class lined up on faces of a 3.5 amino acids/turn helical projection, allowing the construction of a model of the interacting surfaces in which the helices are associated in a left‐handed pentameric coiled‐coil configuration. Molecular modeling simulations (to be described elsewhere in detail) confirm that the helices readily form a left‐handed coiled‐coil helical bundle and have yielded molecular models for the interacting surfaces, the best of which is identical to that predicted by the mutagenesis. Residues lining the pore show considerable structural sensitivity to mutation, indicating that care must be taken in interpreting the results of mutagenesis studies of channels. The cylindrical ion pore (minimal diameter of 2 A) appears to be defined largely by hydrophobic residues (I40, L43 and I47) with only two mildly polar elements contributed by sulfurs in residues C36 and M50.

Calmodulin regulates ryanodine receptor-mediated Ca(2+) release through a conserved binding site. The crystal structure of Ca(2+)-calmodulin bound to this conserved site reveals that calmodulin recognizes two hydrophobic anchor residues at a novel "1-17" spacing that brings the calmodulin lobes close together but prevents them from contacting one another. NMR residual dipolar couplings demonstrate that the detailed structure of each lobe is preserved in solution but also show that the lobes experience domain motions within the complex. FRET measurements confirm the close approach of the lobes in binding the 1-17 target and show that calmodulin binds with one lobe to a peptide lacking the second anchor. We suggest that calmodulin regulates the Ca(2+) channel by switching between the contiguous binding mode seen in our crystal structure and a state where one lobe of calmodulin contacts the conserved binding site while the other interacts with a noncontiguous site on the channel.

Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time glutathione dynamics in living cells. Using RT, we observe enhanced antioxidant capability of activated neurons and dynamic glutathione changes during ferroptosis. RT is thus a versatile tool that can be used for both confocal microscopy and flow cytometry based high-throughput quantification of glutathione levels in single cells. We envision that this new glutathione probe will enable opportunities to study glutathione dynamics and transportation and expand our understanding of the physiological and pathological roles of glutathione in living cells.

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We present the theory of site-directed dichroism and its application to the determination of rotational and orientational constraints for oriented polypeptides such as transmembrane helices. Infrared spectroscopy dichroism measurements of single amide I vibrational modes corresponding to 13C-labeled sites within the polypeptide contain information about the helix tilt and rotation angles. This information is readily extracted by analysis of the dichroism of a set of sites along the peptide sequence. Data for just two consecutive sites in the dimeric transmembrane domain of glycophorin A yield the tilt of the helix axis with respect the membrane normal and the rotation of the helix about its axis. By using dichroism data from three consecutive sites, the helix orientation parameters and the orientation of the amide I transition dipole moment, α, can be obtained; the parameters are in close agreement with the solution NMR structure of the glycophorin A peptide dimer and literature values for α. The approach provides orientational information about selectively labeled peptides even under conditions of modest fractional sample order.