Dimerization of Nitrophorin 4 at Low pH and Comparison to the K1A Mutant of Nitrophorin 1

详细信息    查看全文

• 作者：Robert E. Berry ; Fei Yang ; Tatiana K. Shokhireva ; Angela M. Amoia ; Sarah A. Garrett ; Allena M. Goren ; Stephanie R. Korte ; Hongjun Zhang ; Andrzej Weichsel ; William R. Montfort ; F. Ann Walker
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：January 20, 2015
• 年：2015
• 卷：54
• 期：2
• 页码：208-220
• 全文大小：542K
• ISSN：1520-4995

文摘

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 K_d of 鈭? 渭M. 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鈥揹imer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and ¹H{¹⁵N} 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 鈥渃losed loop鈥?form of the A鈥揃 and G鈥揌 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.