title: β-Turn sequences promote stability of peptide substrates for kinases within the cytosolic environment.
abstract: A strategy was developed to extend the lifetime of an peptide-based substrate for Abl kinase in the cytosolic environment. Small β-turn structures were added to the peptide's N-terminus to block entry into peptidase catalytic sites. The influence of the size of the β-turn and two covalent cross-linking strategies on the rate of hydrolysis was assessed. The most peptidase-resistant substrate was degraded at a rate of 0.6 pmol mg(-1) s(-1) and possessed a half-life of 20.3 ± 1.7 min in a Baf/BCR-ABL cytosolic lysate, representing 16- and 40-fold improvements, respectively, over that of a control peptide lacking the β-turn structure. Furthermore, the kcat/KM value of this peptide was 432 μM(-1) min(-1), a 1.25× increase over the unmodified control, verifying that the added β-turn did not hinder the substrate properties of the peptide. This improved peptide was microinjected into single Baf/BCR-ABL cells and substrate phosphorylation measured. Zero to forty percent of the peptide was phosphorylated in the single cells. In contrast, when the control peptide without a β-turn was loaded into cells, the peptide was too rapidly degraded to detect phosphorylation. This work demonstrates that small β-turn structures can render peptides more resistant to hydrolysis while retaining substrate efficacy and shows that these stabilized peptides have the potential to be of high utility in single-cell enzyme assays.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Shan
authors.initials: S
authors.lastname: Yang
authors.forename: Angela
authors.initials: A
authors.lastname: Proctor
authors.forename: Lauren L
authors.initials: LL
authors.lastname: Cline
authors.forename: Kaiulani M
authors.initials: KM
authors.lastname: Houston
authors.forename: Marcey L
authors.initials: ML
authors.lastname: Waters
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003600:Cytosol / Q000737:chemistry* / Q000201:enzymology*; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D016044:Fusion Proteins, bcr-abl