title: Mass spectrometric characterization of oligomers in Pseudomonas aeruginosa azurin solutions.
abstract: We have employed laser-induced liquid bead ion desorption mass spectroscopy (LILBID MS) to study the solution behavior of Pseudomonas aeruginosa azurin as well as two mutants and corresponding Re-labeled derivatives containing a Re(CO)(3)(4,7-dimethyl-1,10-phenanthroline)(+) chromophore appended to a surface histidine. LILBID spectra show broad oligomer distributions whose particular patterns depend on the solution composition (pure H(2)O, 20-30 mM NaCl, 20 and 50 mM NaP(i) or NH(4)P(i) at pH = 7). The distribution maximum shifts to smaller oligomers upon decreasing the azurin concentration and increasing the buffer concentration. Oligomerization is less extensive for native azurin than its mutants. The oligomerization propensities of unlabeled and Re-labeled proteins are generally comparable, and only Re126 shows some preference for the dimer that persists even in highly diluted solutions. Peak shifts to higher masses and broadening in 20-50 mM NaP(i) confirm strong azurin association with buffer ions and solvation. We have found that LILBID MS reveals the solution behavior of weakly bound nonspecific protein oligomers, clearly distinguishing individual components of the oligomer distribution. Independently, average data on oligomerization and the dependence on solution composition were obtained by time-resolved anisotropy of the Re-label photoluminescence that confirmed relatively long rotation correlation times, 6-30 ns, depending on Re-azurin and solution composition. Labeling proteins with Re-chromophores that have long-lived phosphorescence extends the time scale of anisotropy measurements to hundreds of nanoseconds, thereby opening the way for investigations of large oligomers with long rotation times.
authors: Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany.
authors: Lucie
authors: L
authors: Sokolová
authors: Heather
authors: H
authors: Williamson
authors: Jan
authors: J
authors: Sýkora
authors: Martin
authors: M
authors: Hof
authors: Harry B
authors: HB
authors: Gray
authors: Bernd
authors: B
authors: Brutschy
authors: Antonín
authors: A
authors: Vlcek
mesh_terms: D001400:Azurin / Q000737:chemistry*; D056831:Coordination Complexes / Q000737:chemistry; D013058:Mass Spectrometry; D010618:Phenanthrolines / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D012211:Rhenium / Q000737:chemistry; D012996:Solutions / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
chemical_list: D056831:Coordination Complexes; D010618:Phenanthrolines; D011108:Polymers; D012996:Solutions; D001400:Azurin; D012211:Rhenium; C025205:1,10-phenanthroline