Amyloid-β (Aβ) is a 39–42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimer’s disease (AD). The most prominent forms of Aβ are Aβ1–40 and Aβ1–42, which differ by two amino acids (I and A) at the C-terminus. However, Aβ42 is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of AβM01–42 amyloid fibrils derived from over 500 13C−13C, 13C−15N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3) shows that the fibril core consists of a dimer of Aβ42 molecules, each containing four β-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular 13C−15N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15–A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aβ42 aggregation.

Conspectus During the three decades 1980–2010, magic angle spinning (MAS) NMR developed into the method of choice to examine many chemical, physical and biological problems. In particular, a variety of dipolar recoupling methods to measure distances and torsion angles can now constrain molecular structures to high resolution. However, applications are often limited by the low sensitivity of the experiments, due in large part to the necessity of observing spectra of low-γ nuclei such as the I = ½ species 13C or 15N. The difficulty is still greater when quadrupolar nuclei, like 17O or 27Al, are involved. This problem has stimulated efforts to increase the sensitivity of MAS experiments. A particularly powerful approach is dynamic nuclear polarization (DNP) which takes advantage of the higher equilibrium polarization of electrons (which conventionally manifests in the great sensitivity advantage of EPR over NMR). In DNP, the sample is doped with a stable paramagnetic polarizing agent and irradiated with microwaves to transfer the high polarization in the electron spin reservoir to the nuclei of interest. The idea was first explored by Overhauser and Slichter in 1953. However, these experiments were carried out on static samples, at magnetic fields that are low by current standards. To be implemented in contemporary MAS NMR experiments, DNP requires microwave sources operating in the subterahertz regime — roughly 150–660 GHz — and cryogenic MAS probes. In addition, improvements were required in the polarizing agents, because the high concentrations of conventional radicals that are required to produce significant enhancements compromise spectral resolution. In the last two decades scientific and technical advances have addressed these problems and brought DNP to the point where it is achieving wide applicability. These advances include the development of high frequency gyrotron microwave sources operating in the subterahertz frequency range. In addition, low temperature MAS probes were developed that permit in-situ microwave irradiation of the samples. And, finally, biradical polarizing agents were developed that increased the efficiency of DNP experiments by factors of ~4 at considerably lower paramagnet concentrations. Collectively these developments have made it possible to apply DNP on a routine basis to a number of different scientific endeavors, most prominently in the biological and material sciences. This Account reviews these developments, including the primary mechanisms used to transfer polarization in high frequency DNP, and the current choice of microwave sources and biradical polarizing agents. In addition, we illustrate the utility of the technique with a description of applications to membrane and amyloid proteins that emphasizes the unique structural information that is available in these two cases.

We report a magic angle spinning (MAS) NMR structure of the drug-resistant S31N mutation of M218–60 from Influenza A. The protein was dispersed in diphytanoyl-sn-glycero-3-phosphocholine lipid bilayers, and the spectra and an extensive set of constraints indicate that M218–60 consists of a dimer of dimers. In particular, ~280 structural constraints were obtained using dipole recoupling experiments that yielded well-resolved 13C–15N, 13C–13C, and 1H–15N 2D, 3D, and 4D MAS spectra, all of which show cross-peak doubling. Interhelical distances were measured using mixed 15N/13C labeling and with deuterated protein, MAS at ωr/2π = 60 kHz, ω0H/2π = 1000 MHz, and 1H detection of methyl–methyl contacts. The experiments reveal a compact structure consisting of a tetramer composed of four transmembrane helices, in which two opposing helices are displaced and rotated in the direction of the membrane normal relative to a four-fold symmetric arrangement, yielding a two-fold symmetric structure. Side chain conformations of the important gating and pH-sensing residues W41 and H37 are found to differ markedly from four-fold symmetry. The rmsd of the structure is 0.7 Å for backbone heavy atoms and 1.1 Å for all heavy atoms. This two-fold symmetric structure is different from all of the previous structures of M2, many of which were determined in detergent and/or with shorter constructs that are not fully active. The structure has implications for the mechanism of H+ transport since the distance between His and Trp residues on different helices is found to be short. The structure also exhibits two-fold symmetry in the vicinity of the binding site of adamantyl inhibitors, and steric constraints may explain the mechanism of the drug-resistant S31N mutation.

The serine/threonine kinase protein kinase B (PKB)/ Akt is a critical regulator of insulin signaling, cell survival, and oncogenesis. The activation mechanisms of this key kinase are well characterized. In contrast, inactivation of PKB signaling by phosphatases is less well understood. To study the dynamics of PKB signaling in live cells, we generated a genetically encoded fluorescent reporter for PKB activity that reversibly responds to stimuli activating phosphatidylinositol 3-kinase. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer, allowing real-time imaging of phosphorylation catalyzed by PKB. Because of its reversibility, the reporter also allows termination of PKB signaling by phosphatases to be monitored. We found that PKB signaling in the cytosol was more rapid and more transient compared with that in the nucleus, suggesting the presence of differentially regulated phosphatase activity in these two compartments. Furthermore, targeting of the reporter to the plasma membrane, where PKB is activated, resulted in accelerated and prolonged response compared with the response in the cytosol, suggesting that release of PKB or its substrates from the membrane is required for desensitization of PKB signaling. These data reveal spatio-temporal gradients of both signal propagation and signal termination in PKB signaling. Protein kinase B (PKB)1 /Akt is a serine/threonine kinase that is the prominent mediator of pathways resulting in enhanced cell growth and cell survival. In 1991 it was identified as the transforming component of AKT8, a virus correlating with a high incidence of spontaneous lymphoma in mice, and named Akt (1). In the same year, two other groups isolated the same kinase via its homology to protein kinase A (PKA) and protein kinase C (PKC) and designated it Rac (related to the A and C kinases) (2) and PKB (3). There are three isozymes in mammals: Akt1/PKB␣, Akt2/PKB␤, and Akt3/PKB␥. While the kinase is referred to by these two names, PKB and Akt, herein it will be referred to as PKB.PKB contains an NH 2 -terminal pleckstrin homology (PH) domain followed by a kinase domain and a short COOH-terminal regulatory tail containing an activating phosphorylation site (4). PKB is activated by recruitment to membranes following stimulation of growth factor receptors. Briefly, activated growth factor receptors lead to plasma membrane recruitment of phosphatidylinositol 3-kinase (PI 3-kinase), which leads to the production of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) at the plasma membrane. The PH domain of PKB binds to the newly formed PIP 3 resulting in translocation of the kinase to the plasma membrane (5-8). Once at the membrane, PKB becomes activated via two sequential phosphorylation steps, first by its upstream kinase PDK-1 on Thr 308 within its activation loop, and next via autophosphorylation at Ser 473 within the COOH-terminal hydrophobic motif (9 -12). PKB is maximally active when phosphorylated at both regulat...

Melanin is ubiquitous in living organisms across different biological kingdoms of life, making it an important, natural biomaterial. Its presence in nature from microorganisms to higher animals and plants is attributed to the many functions of melanin, including pigmentation, radical scavenging, radiation protection, and thermal regulation. Generally, melanin is classified into five typeseumelanin, pheomelanin, neuromelanin, allomelanin, and pyomelaninbased on the various chemical precursors used in their biosynthesis. Despite its long history of study, the exact chemical makeup of melanin remains unclear, and it moreover has an inherent diversity and complexity of chemical structure, likely including many functions and properties that remain to be identified. Synthetic mimics have begun to play a broader role in unraveling structure and function relationships of natural melanins. In the past decade, polydopamine, which has served as the conventional form of synthetic eumelanin, has dominated the literature on melaninbased materials, while the synthetic analogues of other melanins have received far less attention. In this perspective, we will discuss the synthesis of melanin materials with a special focus beyond polydopamine. We will emphasize efforts to elucidate biosynthetic pathways and structural characterization approaches that can be harnessed to interrogate specific structure−function relationships, including electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. We believe that this timely Perspective will introduce this class of biopolymer to the broader chemistry community, where we hope to stimulate new opportunities in novel, melanin-based poly-functional synthetic materials.

A high-resolution map of human phosphorylation networks was constructed by integrating experimentally determined kinase-substrate relationships with other resources, such as in vivo phosphorylation sites.

Many protein kinases are key nodal signaling molecules that regulate a wide range of cellular functions. These functions may require complex spatiotemporal regulation of kinase activities. Here, we show that Protein Kinase A (PKA), Ca2+ and cAMP oscillate in sync in insulin-secreting MIN6 β cells, forming a highly integrated oscillatory circuit. We found that PKA activity was essential for this oscillatory circuit, and was capable of not only initiating the signaling oscillations but also modulating their frequency, thereby diversifying the spatiotemporal control of downstream signaling. Our findings suggest that exquisite temporal control of kinase activity, mediated via signaling circuits resulting from cross-regulation of signaling pathways, can encode diverse inputs into temporal parameters such as oscillation frequency, which in turn contributes to proper regulation of complex cellular functions in a context-dependent manner.

Understanding how specific cAMP signals are organized and relayed to their effectors in different compartments of the cell to achieve functional specificity requires molecular tools that allow precise manipulation of cAMP in these compartments. Here we characterize a new method using bicarbonate-activatable and genetically targetable soluble adenylyl cyclase (sAC) to control the location, kinetics and magnitude of the cAMP signal. Using this live-cell cAMP manipulation in conjunction with fluorescence imaging and mechanistic modeling, we uncover the activation of a resident pool of PKA holoenzyme in the nuclei of HEK-293 cells, modifying the existing dogma of cAMP-PKA signaling in the nucleus. Furthermore, we show that phosphodiesterases (PDE) and A-Kinase Anchoring Proteins (AKAP) are critical in shaping nuclear PKA responses. Collectively, our data suggests a new model where AKAP-localized PDEs tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.