Development of a protease-resistant reporter to quantify BCR-ABL activity in intact cells.

A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids. After an iterative design process, the lead, or designed, peptide X-A possesses a six-fold longer life in a cytosolic lysate than that of the starting peptide. The catalytic efficiency (kcat/KM) of purified ABL kinase for the lead peptide (125 s-1 µM-1) is similar to that of the starting peptide (103 s-1 µM-1) demonstrating preservation of the peptide's ability to serve as a kinase substrate. When incubated in cytosolic lysates, the lead peptide is slowly degraded into 4 fragments over time. In contrast, when loaded into intact cells, the peptide is metabolized into 5 fragments, with only 2 of the fragments corresponding to those in the lysate. Thus the two environments possess differing peptidase activities, which must be accounted for when designing peptidase-resistant peptides. In both settings, the substrate is phosphorylated by BCR-ABL providing a readout of BCR-ABL activity. A small panel of tyrosine kinase inhibitors verified the substrate's specificity for BCR-ABL/ABL kinase activity in both lysates and cells in spite of the multitude of other kinases present. The designed peptide X-A acts as a long-lived BCR-ABL kinase reporter in the leukemic cells possessing the BCR-ABL mutation.

Separation of peptide fragments of a protein kinase C substrate fused to a ß-hairpin by capillary electrophoresis.

Synthetic peptides incorporating well-folded ß-hairpin peptides possess advantages in a variety of cell biology applications by virtue of increased resistance to proteolytic degradation. In this study, the WKpG ß-hairpin peptide fused to a protein kinase C (PKC) substrate was synthesized, and capillary-electrophoretic separation conditions for this peptide and its proteolytic fragments were developed. Fragments of WKpG-PKC were generated by enzymatic treatment with trypsin and Pronase E to produce standards for identification of degradation fragments in a cellular lysate. A simple buffer system of 250 mM H3PO4, pH 1.5 enabled separation of WKpG-PKC and its fragments by capillary electrophoresis in less than 16 min. Using a cellular lysate produced from Ba/F3 cells, the ß-hairpin-conjugated substrate and its PKCα-phosphorylated product could be detected and separated from peptidase-generated fragments produced in a cell lysate. The method has potential application for identification and quantification of WKpG-PKC and its fragments in complex biological systems when the peptide is used as a reporter to assay PKC activity.

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes.

α-Synuclein, an intrinsically-disordered protein associated with Parkinson's disease, interacts with mitochondria, but the details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles by using fluorescence anisotropy and (19)F nuclear magnetic resonance. Both proteins interact strongly with large unilamellar vesicles whose composition is similar to that of the inner mitochondrial membrane, which contains cardiolipin. However, the proteins have no affinity for vesicles mimicking the outer mitochondrial membrane, which lacks cardiolipin. The (19)F data show that the interaction involves α-synuclein's N-terminal region. These data indicate that the middle of the N-terminal region, which contains the KAKEGVVAAAE repeats, is involved in binding, probably via electrostatic interactions between the lysines and cardiolipin. We also found that the strength of α-synuclein binding depends on the nature of the cardiolipin acyl side chains. Eliminating one double bond increases affinity, while complete saturation dramatically decreases affinity. Increasing the temperature increases the binding of wild-type, but not the A30P variant. The data are interpreted in terms of the properties of the protein, cardiolipin demixing within the vesicles upon binding of α-synuclein, and packing density. The results advance our understanding of α-synuclein's interaction with mitochondrial membranes.

Interaction of α-synuclein and a cell penetrating fusion peptide with higher eukaryotic cell membranes assessed by ¹9F NMR.

We show that fluorine NMR can be used to monitor the insertion and change in conformation of a ¹9F-labeled cell-penetrating peptide upon interacting with the cellular plasma membrane. α-Synuclein and a construct comprising a cell-penetrating peptide covalently attached to its N-terminus were studied. Important information about the interaction of the proteins with CHO-K1 cells was obtained by monitoring the diminution of ¹9F resonances of 3-fluoro-l-tyrosine labeled proteins. For α-synuclein, a decrease in the resonance from position 39 was observed indicating that only the N-terminal third region of the protein interacts with plasma membrane. However, when the fusion construct was incubated with the cells, a decrease in the resonance from the fusion peptide region was noted with no change in the resonances from α-synuclein region. Longer incubation, studied by using confocal fluorescence microscopy, revealed that the fusion construct translocates into the cells, but α-synuclein alone did not cross the membrane in significant amounts.

Protein nuclear magnetic resonance under physiological conditions.

Almost everything we know about protein biophysics comes from studies on purified proteins in dilute solution. Most proteins, however, operate inside cells where the concentration of macromolecules can be >300 mg/mL. Although reductionism-based approaches have served protein science well for more than a century, biochemists now have the tools to study proteins under these more physiologically relevant conditions. We review a part of this burgeoning postreductionist landscape by focusing on high-resolution protein nuclear magnetic resonance (NMR) spectroscopy, the only method that provides atomic-level information over an entire protein under the crowded conditions found in cells.

Alpha-Synuclein conformation affects its tyrosine-dependent oxidative aggregation.

Oxidative stress and aggregation of the protein alpha-synuclein are thought to be key factors in Parkinson's disease. Previous work shows that cytochrome c with H(2)O(2) causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including alpha-synuclein. Here, we examine the role of each of alpha-synuclein's four tyrosine residues and how the protein's conformation affects covalent oxidative aggregation. When alpha-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild-type-like covalent aggregation in the presence of a denaturant or when alpha-synuclein is present in noncovalent fibrils. We also show that preformed oxidative aggregates are not incorporated into noncovalent fibrils. These data provide insight into how dityrosine may be formed in Lewy bodies seen in Parkinson's disease.