Chiral stapled o -OPEs show excellent circular polarized luminescence responses ( g _lum of 1.1 × 10 ⁻² ) which can be modulated by carbophilic interactions.

The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation.

Na⁺-H⁺ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including Na–phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser⁴⁶, Ser¹⁶², Ser¹⁸¹, Ser²⁶⁹, Ser²⁸⁰, Ser²⁹¹, Thr²⁹³, Ser²⁹⁹, and Ser³⁰² did not affect phosphate uptake, but Ser²⁹⁰Ala substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser²⁹⁰ was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser²⁹⁰. Mutating ²⁵⁷VPF²⁵⁹ eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser²⁹⁰ dephosphorylation of recombinant NHERF1. Hydrogen–deuterium exchange MS revealed that β-sheets in NHERF1’s PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser²⁹⁰ rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein–coupled receptor kinase 6A controls the Ser²⁹⁰ phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.