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首页> 外文期刊>The journal of physical chemistry, B. Condensed matter, materials, surfaces, interfaces & biophysical >Mass Spectrometric Characterization of Oligomers in Pseudomonas aeruginosa Azurin Solutions
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Mass Spectrometric Characterization of Oligomers in Pseudomonas aeruginosa Azurin Solutions

机译：铜绿假单胞菌天青素溶液中低聚物的质谱表征

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摘要

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)~+ chromo-phore 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 NaP_i or NH4P_i at pH = 7). The distribution maximum shifts to smaller oligomers upon decreasing theazurin 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 Rel26 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.
机译：我们已采用激光诱导的液珠离子解吸质谱（LILBID MS）来研究铜绿假单胞菌铜绿假单胞菌以及两个突变体和相应的含有Re（CO）3（4,7-dimethyl- 1,10-菲咯啉）〜+荧光团附加在组氨酸表面LILBID光谱显示了广泛的低聚物分布，其特定模式取决于溶液的组成（纯水，pH = 7的20-30 mM NaCl，20和50 mM NaP_1或NH4P_1）。降低天青素浓度并增加缓冲液浓度后，分布最大值转移至较小的低聚物。天然天青蛋白的低聚作用不如其突变体广泛。未标记和重新标记的蛋白质的低聚倾向通常是可比的，只有Rel26偏爱即使在高度稀释的溶液中仍然存在的二聚体。峰移至更高质量并在20-50 mM NaP_i中加宽证实了天青蛋白与缓冲离子和溶剂化的强相关性。我们发现LILBID MS揭示了弱结合的非特异性蛋白质寡聚物的溶液行为，清楚地区分了寡聚物分布的各个组成部分。独立地，通过Re-标签光致发光的时间分辨各向异性获得有关低聚反应和对溶液组成的依赖性的平均数据，该时间证实了相对较长的旋转相关时间（6-30 ns），具体取决于Re-天青素和溶液组成。用具有长寿命磷光的Re-发色团标记蛋白质可以将各向异性测量的时间范围扩展到数百纳秒，从而为研究具有长旋转时间的大型寡聚物开辟了道路。

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