A "spindle and thread" mechanism unblocks p53 translation by modulating N-terminal disorder.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.

Gas-Phase Collisions with Trimethylamine-N-Oxide Enable Activation-Controlled Protein Ion Charge Reduction.

Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35 kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation. ᅟ.

Alzheimer's disease and cigarette smoke components: effects of nicotine, PAHs, and Cd(II), Cr(III), Pb(II), Pb(IV) ions on amyloid-ß peptide aggregation.

Cigarette smoking is a significant risk factor for Alzheimer's disease (AD), which is associated with extracellular brain deposits of amyloid plaques containing aggregated amyloid-ß (Aß) peptides. Aß aggregation occurs via multiple pathways that can be influenced by various compounds. Here, we used AFM imaging and NMR, fluorescence, and mass spectrometry to monitor in vitro how Aß aggregation is affected by the cigarette-related compounds nicotine, polycyclic aromatic hydrocarbons (PAHs) with one to five aromatic rings, and the metal ions Cd(II), Cr(III), Pb(II), and Pb(IV). All PAHs and metal ions modulated the Aß aggregation process. Cd(II), Cr(III), and Pb(II) ions displayed general electrostatic interactions with Aß, whereas Pb(IV) ions showed specific transient binding coordination to the N-terminal Aß segment. Thus, Pb(IV) ions are especially prone to interact with Aß and affect its aggregation. While Pb(IV) ions affected mainly Aß dimer and trimer formation, hydrophobic toluene mainly affected formation of larger aggregates such as tetramers. The uncharged and hydrophilic nicotine molecule showed no direct interactions with Aß, nor did it affect Aß aggregation. Our Aß interaction results suggest a molecular rationale for the higher AD prevalence among smokers, and indicate that certain forms of lead in particular may constitute an environmental risk factor for AD.