Berry Robert E, Yang Fei, Shokhireva Tatiana K, Amoia Angela M, Garrett Sarah A, Goren Allena M, Korte Stephanie R, Zhang Hongjun, Weichsel Andrzej, Montfort William R, Walker F Ann
Department of Chemistry and Biochemistry, The University of Arizona , P.O. Box 210041, Tucson, Arizona 85721-0041, United States.
Biochemistry. 2015 Jan 20;54(2):208-20. doi: 10.1021/bi5013047. Epub 2014 Dec 23.
Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer-dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and (1)H{(15)N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The "closed loop" form of the A-B and G-H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.
嗜唾蛋白4是来自南美锥蝽唾液腺的四种携带一氧化氮的血红素蛋白之一，在pH 5.0时形成同二聚体，解离常数Kd约为8 μM。该二聚体在pH 5.5时开始解离，在pH 7.3时完全解离为单体，即使在3.7 mM时也是如此。通过将一氧化氮与血红素结合，二聚体得到显著稳定，在pH 7.3时，需要稀释至远低于0.2 mM才能使NP4-NO同二聚体完全解离。用于研究同二聚体和单体-二聚体平衡的主要技术是在pH 5.0时进行尺寸排阻快速蛋白质液相色谱，以及作为pH和浓度函数的(1)H{(15)N}异核单量子相干光谱。NP4定点突变体（A1K、D30A、D30N、V36A/D129A/L130A、K38A、R39A、K125A、K125E、D132A、L133V和K38Q/R39Q/K125Q）的制备表明，N端、D30、D129、D132、至少一个血红素丙酸酯，以及与之相关的E32和D35可能也参与了二聚化。单体NP4的A-B和G-H柔性环的“闭环”形式在pH 5.6时报道的单体蛋白晶体结构中占主导，但在pH 7.5时则不然，且涉及除D132外上述所有残基，是二聚体形成所必需的。野生型NP1不形成同二聚体，但NP1(K1A)和天然N端NP1在一氧化氮存在下形成二聚体。然而，在没有一氧化氮的情况下，NP1的同二聚体稳定性远低于NP4。这表明NP4 C端区域中存在但NP1中不存在的额外天冬氨酸或谷氨酸残基也参与了二聚体的稳定。
