The crystal structure of IB␣ in complex with the transcription factor, nuclear factor -B (NF-B) shows six ankyrin repeats, which are all ordered. Electron density was not observed for most of the residues within the PEST sequence, although it is required for high-affinity binding. To characterize the folded state of IB␣ (67-317) when it is not in complex with NF-B, we have carried out circular dichroism (CD) spectroscopy, 8-anilino-1-napthalenesulphonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange experiments. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amount of ANS-binding and amide exchange suggest that the protein may have molten globule character. The amide exchange experiments show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. IB␣ unfolds with a T m of 42°C, and forms a soluble aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biological function of IB␣ are discussed.

SUMMARY Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO3−. The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into 3-dimensional collagen matrix.

SUMMARYIκBα is an ankyrin repeat protein that inhibits NF-κB transcriptional activity by sequestering NF-κB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IκBα ankyrin repeat domain to NF-κB(p50/p65) using surface plasmon (SPR) resonance and isothermal titration calorimetry (ITC). SPR data showed that the IκBα and NF-κB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K D.obs of approximately 40 pM at 37 °C. As reported previously, the amino-terminal/DNA binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high affinity binding. This was surprising given the small size of the binding interface formed by this part of the p65. The NF-κB(p50/p65) heterodimer and p65 homodimer bound IκBα with almost indistinguishable thermodynamics except that the NF-κB p65 homodimer was characterized by a more favorable ΔH obs relative to the NF-κB (p50p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (ΔC P,obs ), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be readily accounted for by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.

Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can enable the target protein to fold free from interference. Using both naturally-occurring and artificial polypeptides we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have low sequence complexity and high net charge, but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently-used fusions such as maltose-binding-protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physio-chemical properties they did not perturb the structure, conformational stability nor function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically-disordered fusions, and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.