Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany.
J Phys Chem B. 2011 Apr 28;115(16):4790-800. doi: 10.1021/jp110460k. Epub 2011 Mar 31.
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.
我们采用激光诱导液珠离子解吸质谱（LILBID MS）研究了铜绿假单胞菌天青蛋白及其两个突变体的溶液行为，以及相应的 Re 标记衍生物。该衍生物通过表面组氨酸连接了一个 Re（CO）（3）（4,7-二甲基-1,10-菲咯啉）（+）发色团。LILBID 光谱显示出广泛的寡聚物分布，其特定模式取决于溶液组成（纯 H2O、20-30 mM NaCl、20 和 50 mM NaP(i)或 NH4P(i)，pH = 7）。随着天青蛋白浓度降低和缓冲液浓度增加，分布最大值向较小的寡聚物转移。与突变体相比，天然天青蛋白的寡聚化程度较低。未标记和 Re 标记蛋白的寡聚化倾向通常相当，只有 Re126 对二聚体表现出一些偏好，这种偏好甚至在高度稀释的溶液中仍然存在。在 20-50 mM NaP(i)中，峰向更高质量移动和变宽证实了天青蛋白与缓冲离子和溶剂化的强烈结合。我们发现，LILBID MS 揭示了弱结合非特异性蛋白质寡聚物的溶液行为，清晰地区分了寡聚物分布的各个组成部分。独立地，通过 Re 标记光致发光的时间分辨各向异性获得了关于寡聚化和对溶液组成依赖性的平均数据，该数据证实了相对较长的旋转相关时间，6-30 ns，这取决于 Re-天青蛋白和溶液组成。用具有长磷光寿命的 Re 发色团标记蛋白质将各向异性测量的时间尺度扩展到数百纳秒，从而为具有长旋转时间的大寡聚物的研究开辟了道路。
