Porter J, Dit Fouque K Jeanne, Miksovska J, Fernandez-Lima F
Department of Chemistry and Biochemistry, Florida International University Miami FL 33199 USA
Biomolecular Science Institute, Florida International University Miami FL 33199 USA.
RSC Adv. 2020 Sep 11;10(56):33861-33867. doi: 10.1039/d0ra04956e. eCollection 2020 Sep 10.
In this work, a proteolytic digest of cytochrome c (microperoxidase 11, MP-11) was used as a model to study the structural aspects of heme protein interactions and porphyrin networks. The MP-11 structural heterogeneity was studied as a function of the starting pH (, pH 3.1-6.1) and concentration (, 1-50 μM) conditions and adduct coordination. Trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) showed the MP-11 structural dependence of the charge state distribution and molecular ion forms with the starting pH conditions. The singly charged (, [M], [M - 2H + NH], [M - H + Na] and [M - H + K]) and doubly charged (, [M + H], [M - H + NH], [M + Na] and [M + K]) molecular ion forms were observed for all solvent conditions, although the structural heterogeneity (, number of mobility bands) significantly varied with the pH value and ion form. The MP-11 dimer formation as a model for heme-protein protein interactions showed that dimer formation is favored toward more neutral pH and favored when assisted by salt bridges (, NH , Na and K H). Inspection of the dimer mobility profiles (2+ and 3+ charge states) showed a high degree of structural heterogeneity as a function of the solution pH and ion form; the observation of common mobility bands suggest that the different salt bridges can stabilize similar structural motifs. In addition, the salt bridge influence on the MP-11 dimer formations was measured using collision induced dissociation and showed a strong dependence with the type of salt bridge (, a CE of 10.0, 11.5, 11.8 and 13.0 eV was observed for [2M + H], [2M - H + NH], [2M + Na] and [2M + K], respectively). Measurements of the dimer equilibrium constant showed that the salt bridge interactions increase the binding strength of the dimeric species.
在这项工作中，细胞色素c的蛋白水解消化产物（微过氧化物酶11，MP-11）被用作模型，以研究血红素蛋白相互作用和卟啉网络的结构方面。研究了MP-11的结构异质性与起始pH值（pH 3.1 - 6.1）、浓度（1 - 50 μM）条件以及加合物配位的关系。阱式离子淌度光谱联用质谱（TIMS-MS）显示了MP-11的电荷态分布和分子离子形式对起始pH条件的结构依赖性。在所有溶剂条件下均观察到单电荷（如[M]、[M - 2H + NH]、[M - H + Na]和[M - H + K]）和双电荷（如[M + H]、[M - H + NH]、[M + Na]和[M + K]）分子离子形式，尽管结构异质性（淌度带数量）随pH值和离子形式有显著变化。作为血红素 - 蛋白质相互作用模型的MP-11二聚体形成表明，二聚体形成更倾向于在更接近中性的pH条件下，并且在盐桥（如NH、Na和K H）辅助下更有利。对二聚体淌度谱（2 +和3 +电荷态）的检查显示，作为溶液pH和离子形式的函数，存在高度的结构异质性；对共同淌度带的观察表明，不同的盐桥可以稳定相似的结构基序。此外，使用碰撞诱导解离测量了盐桥对MP-11二聚体形成的影响，结果表明其对盐桥类型有很强的依赖性（例如，对于[2M + H]、[2M - H + NH]、[2M + Na]和[2M + K]，分别观察到的碰撞能量为10.0、11.5、11.8和13.0 eV）。二聚体平衡常数的测量表明，盐桥相互作用增加了二聚体物种的结合强度。
