A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.

A common single-nucleotide polymorphism in the human brain-derived neurotrophic factor (BDNF) gene results in a Val66Met substitution in the BDNF prodomain region. This single-nucleotide polymorphism is associated with alterations in memory and with enhanced risk to develop depression and anxiety disorders in humans. Here we show that the isolated BDNF prodomain is detected in the hippocampus and that it can be secreted from neurons in an activity-dependent manner. Using nuclear magnetic resonance spectroscopy and circular dichroism we find that the prodomain is intrinsically disordered, and the Val66Met substitution induces structural changes. Surprisingly, application of Met66 (but not Val66) BDNF prodomain induces acute growth cone retraction and a decrease in Rac activity in hippocampal neurons. Expression of p75NTR and differential engagement of the Met66 prodomain to the SorCS2 receptor are required for this effect. These results identify the Met66 prodomain as a new active ligand which modulates neuronal morphology.

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BACKGROUND AND PURPOSECardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age-related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS-31 also protected the electron carrier function of cytochrome c. EXPERIMENTAL APPROACHInteractions of SS-31 with cardiolipin were studied using liposomes and bicelles containing phosphatidylcholine alone or with cardiolipin. Structural interactions were assessed by fluorescence spectroscopy, turbidity and nuclear magnetic resonance. Effects of cardiolipin on electron transfer kinetics of cytochrome c were determined by cytochrome c reduction in vitro and oxygen consumption using mitoplasts, frozen and fresh mitochondria.

SUMMARY The CARMA1/Bcl10/MALT1 (CBM) signalosome mediates antigen receptor-induced NF-κB signaling to regulate multiple lymphocyte functions. While CARMA1 and Bcl10 contain caspase recruitment domains (CARDs), MALT1 is a paracaspase with structural similarity to caspases. Here we show that the reconstituted CBM signalosome is a helical filamentous assembly in which substoichiometric CARMA1 nucleates Bcl10 filaments. Bcl10 filament formation is a highly cooperative process whose threshold is sensitized by oligomerized CARMA1 upon receptor activation. In cells, both cotransfected CARMA1/Bcl10 complex and the endogenous CBM signalosome are filamentous morphologically. Combining crystallography, nuclear magnetic resonance, and electron microscopy, we reveal the structure of the Bcl10 CARD filament and the mode of interaction between CARMA1 and Bcl10. Structure-guided mutagenesis confirmed the observed interfaces in Bcl10 filament assembly and MALT1 activation in vitro and NF-κB activation in cells. These data support a paradigm of nucleation-induced signal transduction with threshold response due to cooperativity and signal amplification by polymerization.

Analysis of the ratio of transverse and longitudinal relaxation rates (R2/R1) is an approach commonly used for estimation of overall correlation time and identification of chemical exchange in biological macromolecules. However, this analysis fails to distinguish between chemical exchange and motional anisotropy. We describe a simple method for identifying chemical exchange and motional anisotropy using the product, R1R2. In the slow tumbling regime, the R1R2 product results in a constant value that is independent of overall correlation time and motional anisotropy. This analysis provides a simple method for rapidly estimating and dissociating the effects of motional anisotropy and chemical exchange in NMR heteronuclear spin relaxation data. We demonstrate the utility of the method with 15N relaxation data collected on the proteins E. coli ribonuclease H and the trimeric E. coli membrane associated lipoprotein lpp.

Two-dimensional 1H NMR spectroscopy and hydrogen exchange methods have been used to characterize the alcohol-denatured state of monellin. Monellin is a sweet tasting protein composed of two chains. In the native state, the A-chain consists entirely of beta-structure, and the B-chain contains both alpha- and beta-structure. Upon addition of either 50% ethanol or 50% trifluoroethanol (TFE), the native structure of monellin is disrupted resulting in an alcohol-denatured state with properties different from those of the random coil state. In the alcohol-denatured state, the far-UV circular dichroism (CD) spectrum displays a higher helical content relative to the native state and the intensity of the near-UV CD signal is completely lost. One-dimensional NMR studies show that there are approximately 14 amide protons protected from exchange with solvent in the alcohol-denatured state and that large portions of the protein exchange at a rate that is comparable to the exchange rate of the protein in urea. Utilizing hydrogen exchange trapping techniques, the slowly exchanging residues are identified at pH 2.0 in 50% ethanol and 50% TFE (A10-A15, A18, A19, A21, A24, and A39) and are found to be clustered on one region of the A-chain. Preliminary 2D NMR assignments show that in the alcohol-denatured state the A-chain of monellin undergoes structural reorganization, with one strand of the native state beta-sheet on the A-chain (residues A17-A30) becoming an alpha-helix in the alcohol-denatured state. The secondary structure of the A-chain in the alcohol-denatured state is different from the native state structure, although the slowly exchanging residues are similar.

Summary Antigen recognition of peptide-major histocompatibility complexes (pMHCs) by T-cells, a key step in initiating adaptive immune responses, is performed by the T-cell receptor (TCR) bound to CD3 heterodimers. However, the biophysical basis of the transmission of TCR-CD3 extracellular interaction into a productive intracellular signaling sequence remains incomplete. Herein, we used nuclear magnetic resonance (NMR) spectroscopy combined with mutational analysis and computational docking to derive a structural model of the extracellular TCR-CD3 assembly. In the inactivated state, CD3γε interacts with the helix-3 and helix 4-F strand regions of the TCR Cβ subunit while CD3δε interacts with the F and C strand regions of TCR Cα subunit in this model, thereby placing the CD3 subunits on opposing sides of the TCR. Together this work identifies the molecular contacts between the TCR and CD3 subunits thereby identifying a physical basis for transmitting an activating signal through the complex.