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 H2O, 20–30 mM NaCl, 20 and 50 mM NaPi or NH4Pi 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, 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 NaPi confirm strong azurin association with buffer ions and solvation. We have found that LILBID MS reveals the solution behavior of weakly bound nonspecific 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 timescale of anisotropy measurements to hundreds of ns, thereby opening the way for investigations of large oligomers with long rotation times.

Tryptophan-Accelerated Electron Flow Across a Protein–Protein Interface

“…Although other Re-labeled azurins also form oligomers in concentrated solutions, 41 the well-coupled interface is a unique feature of the forward ET in (Re126W122Cu II ) 2 . Crystals of All-Phe Re107Cu II and Re124K122Cu II show two molecules interacting through dmp-dmp (phen-phen in the latter) π-stacking that connects the two Re labels (Figure 9), precluding intermolecular ET due to long Re-//-Cu distances.…”

Phototriggering Electron Flow through ReI‐modified Pseudomonas aeruginosa Azurins

“…[37] Oligomers also are indicated by relatively high values of protein rotation times T 2 that were determined from the biexponential emission anisotropy decay of Re I (dmp)(W)AzCu II in 20 mM NaP i , pH ~7.1 (3.7 mM: T 1 = 5.1±4.3 ns (18%), T 2 = 31.5 (82%); 0.37 mM: T 1 = 5.1±3.4 ns (36%), T 2 = 16.5±3.5 ns (64%)) and Re I (dmp)(Y)AzZn II in 50 mM Na/KP i , D 2 O, pD ~7.1 (4.4 mM: T 1 = 4.8±0.7 ns (32%), T 2 = 50±2 ns (68%); 0.44 mM: T 1 ≅ 1 ns, T 2 = 30±1 ns (45%)). At the same time, the relatively large T 1 values are attributable to slower Re(dmp)-chromophore motions relative to the peptide than those in Re-azurins that do not contain an aromatic amino acid next to the Re label.…”

“…Similar interaction occurs between dmp and F122 (or Y122) in Re I (dmp)(F or Y)AzM. Although azurins, including Re(dmp)(W)AzCu II , form oligomers in solutions, [37] the kinetics data can be interpreted in terms of intramolecular ET and relaxation steps only. Intraprotein ET is much faster than ET across protein-protein interfaces in oligomers, especially when the proteins are bound by nonspecific forces, as indicated [37] by LILBID mass spectra.…”

“…Although azurins, including Re(dmp)(W)AzCu II , form oligomers in solutions, [37] the kinetics data can be interpreted in terms of intramolecular ET and relaxation steps only. Intraprotein ET is much faster than ET across protein-protein interfaces in oligomers, especially when the proteins are bound by nonspecific forces, as indicated [37] by LILBID mass spectra. Intermolecular ET would not be competitive even if the indole-dmp-dmp-indole interaction found in Re(dmp)AzCu II dimers in crystals (Figure 2) were preserved in an oligomer core, as each dmp ligand is coupled to an indole of the same azurin molecule, being separated from that of the second molecule by another Re(dmp) unit.…”

Light-Induced Nanosecond Relaxation Dynamics of Rhenium-Labeled Pseudomonas aeruginosa Azurins

“…25 For some mutants, the "slow" IR relaxation time increases with increasing concentration 25 due to aggregation. 53 Moreover, time-dependent Reluminescence anisotropy exhibits a site-and concentration dependent 100-1300 ps decay attributable to rotation or "wobbling" of the Re chromophore relative to the protein. 25,53 Excited-state (CO) IR bands of photo-ET-active Re(dmp)-tryptophan azurins simultaneously decrease in intensity and shift higher due to concomitant (ultra)fast ET and relaxation motions, respectively.…”