Heterogeneous protein co-assemblies with tunable functional domain stoichiometry.

In nature, the precise heterogeneous co-assembly of different protein domains gives rise to supramolecular machines that perform complex functions through the co-integrated activity of the individual protein subunits. A synthetic approach capable of mimicking this process would afford access to supramolecular machines with new or improved functional capabilities. Here we show that the distinct peptide strands of a heterotrimeric α-helical coiled-coil (i.e., peptides "A", "B", and "C") can be used as fusion tags for heterogeneous co-assembly of proteins into supramolecular structures with tunable subunit stoichiometry. In particular, we demonstrate that recombinant fusion of A with NanoLuc luciferase (NL-A), B with superfolder green fluorescent protein (sfGFP-B), and C with mRuby (mRuby-C) enables formation of ternary complexes capable of simultaneously emitting blue, green, and red light via sequential bioluminescence and fluorescence resonance energy transfer (BRET/FRET). Fusion of galectin-3 onto the C-terminus of NL-A, sfGFP-B, and mRuby-C endows the ternary complexes with lactose-binding affinity that can be tuned by varying the number of galectin-3 domains integrated into the complex from one to three, while maintaining BRET/FRET function. The modular nature of the fusion protein design, the precise control of domain stoichiometry, and the multiplicity afforded by the three-stranded coiled-coil scaffold provides access to a greater range of subunit combinations than what is possible with heterodimeric coiled-coils used previously. We envision that access to this expanded range of co-integrated protein domain diversity will be advantageous for future development of designer supramolecular machines for therapeutic, diagnostic, and biotechnology applications.

Diffuse Large B-Cell Lymphoma Presenting as an Unusual Paraneoplastic Neurologic Syndrome Affecting the Central and Peripheral Nervous Systems.

A 68-year-old man presented with a two-week history of ascending, symmetric, sensory neuropathy concerning an acute inflammatory demyelinating polyneuropathy that briefly responded to intravenous immunoglobulin (IVIg) therapy. The initial workup was negative for acquired causes. After three months of poor response to standard therapies, he was hospitalized for severe disability, unintentional weight loss, and additional, unexplained neurologic symptoms including cerebellar ataxia, dysarthria, and muscle twitching. Positron emission tomography revealed hypermetabolism isolated to the bone marrow. Bone marrow biopsy confirmed the diagnosis of diffuse large B-cell lymphoma (DLBCL). Due to rapidly worsening performance status, plasmapheresis was initiated prior to treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemotherapy. His symptoms initially improved following plasmapheresis and resolved with chemotherapy. One year following treatment, he remains in complete remission. This case describes a unique paraneoplastic neurologic syndrome involving the central and peripheral nervous system that responded well to plasmapheresis and systemic chemotherapy.

Benzocoumarin Hydrazine: A Large Stokes Shift Fluorogenic Sensor for Detecting Carbonyls in Isolated Biomolecules and in Live Cells.

Detection and quantification of biomolecule carbonylation, a critical manifestation of oxidative stress, allows better understanding of associated disease states. Existing approaches for such analyses require further processing of cells and tissues, which leads to loss of both spatial and temporal information about carbonylated biomolecules in cells. Live cell detection of these species requires sensors that are nontoxic, sufficiently reactive with the biocarbonyl in the intracellular milieu, and detectable with commonly available instrumentation. Presented here is a new fluorescent sensor for biomolecule carbonyl detection: a hydrazine derivative of a benzocoumarin, 7-hydrazinyl-4-methyl-2H-benzo[h]chromen-2-one (BzCH), which meets these requirements. This probe is especially well suited for live cell studies. It can be excited by a laser line common to many fluorescence microscopes. The emission maximum of BzCH undergoes a substantial red shift upon hydrazone formation (from ∼430 to ∼550 nm), which is the result of fluorophore disaggregation. Additionally, the hydrazone exhibits an exceptionally large Stokes shift (∼195 nm). The latter properties eliminate self-quenching of the probe and the need to remove unreacted fluorophore for reliable carbonyl detection. Thus, biomolecule carbonylation can be detected and quantified in cells and in cell extracts in a one-step procedure using this probe.

Intramolecular Catalysis of Hydrazone Formation of Aryl-Aldehydes via ortho-Phosphate Proton Exchange.

Bioorthogonal site-specific chemical reaction to label biomolecules in vitro and in living cells is one of the most powerful and convenient tools in chemical biology. A reactive pairs frequently used for chemical conjugation are aldehydes/ketones with hydrazines/hydrazides/hydroxylamines. Although the reaction is generally specific for the two components, even in a cellular environment, the reaction is very slow under physiological conditions. Addition of a phosphate group at the ortho-position of an aromatic aldehyde increases the reaction rate by an order of magnitude and enhances the aqueous solubility of the reagent and the product. We have synthesized phosphate-substituted aldehyde synthetic models to study kinetics of their reactions with hydrazines and hydrazides that contain a fluorophore. This rapid bioorthogonal reaction should therefore be potentially a very useful reaction for routine site-specific chemical ligations to study and image complex cellular processes in biological systems.