Surveying ubiquitin structure by noncovalent attachment of distance constrained bis(crown) ethers.
Selective noncovalent adduct protein probing (SNAPP) mass spectrometry was recently developed to study solution-phase conformations of proteins by exploiting the specific affinity between 18-crown-6 ether (18C6) and lysine side chains. To obtain more detailed information about protein tertiary structure, a novel noncovalent cross-linking reagent with two 18C6 molecules bridged by a covalent phenyl linker (called PBC for phenyl bis-crown) was synthesized. PBC introduces a distance constraint into SNAPP experiments where pairs of lysine side chains that are held in proximity by tertiary structure should be the most favored binding sites. Application of this method to ubiquitin reveals that PBC can bind to one lysine in a monodentate fashion or bind to two lysines via a bidentate interaction. Comparison with 18C6 can be used to reveal the mode of binding. For the native state of ubiquitin, bidentate binding of PBC is not observed. The partially denatured A-state, however, contains a single pair of lysines that are both chemically available and spaced by less than approximately 19 A (the maximum distance spanning the crown ether binding sites in PBC). Collision-induced dissociation and site-directed mutagenesis reveal that the bidentate PBC attaches to K29 and K33, which is in agreement with previous structural data on the A-state of ubiquitin. PBC is shown to be an effective probe of protein structure in SNAPP experiments, although assigning the specific residues to which PBC is attached can be experimentally challenging.
Ly
Tony
T
Department of Chemistry, University of California, Riverside, California 92521, USA.
Liu
Zhenjiu
Z
Pujanauski
Brian G
BG
Sarpong
Richmond
R
Julian
Ryan R
RR
D000818:Animals; D017360:Arabidopsis / Q000737:chemistry; D029681:Arabidopsis Proteins / Q000737:chemistry; D002417:Cattle; D043844:Crown Ethers / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D010647:Phenyl Ethers / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry*
D029681:Arabidopsis Proteins; D043844:Crown Ethers; D010647:Phenyl Ethers; D025801:Ubiquitin; C031538:phenyl ether; C015762:18-crown-6; D008239:Lysine