Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1

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 "Bill" Montfort, F. Ann Walker

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Abstract

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 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 "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.

Original languageEnglish (US)
Pages (from-to)208-220
Number of pages13
JournalBiochemistry
Volume54
Issue number2
DOIs
StatePublished - Jan 20 2015

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Dimerization
Dimers
Heme
Monomers
Hemeproteins
Liquid chromatography
Propionates
Rhodnius
Aspartic Acid
Dilution
nitrophorin
Glutamic Acid
Proteins
Crystal structure
Salivary Glands
Association reactions
Liquid Chromatography
Spectroscopy
Spectrum Analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Berry, R. E., Yang, F., Shokhireva, T. K., Amoia, A. M., Garrett, S. A., Goren, A. M., ... Walker, F. A. (2015). Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1. Biochemistry, 54(2), 208-220. https://doi.org/10.1021/bi5013047

Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1. / 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 "Bill"; Walker, F. Ann.

In: Biochemistry, Vol. 54, No. 2, 20.01.2015, p. 208-220.

Research output: Contribution to journalArticle

Berry, RE, Yang, F, Shokhireva, TK, Amoia, AM, Garrett, SA, Goren, AM, Korte, SR, Zhang, H, Weichsel, A, Montfort, WB & Walker, FA 2015, 'Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1', Biochemistry, vol. 54, no. 2, pp. 208-220. https://doi.org/10.1021/bi5013047
Berry RE, Yang F, Shokhireva TK, Amoia AM, Garrett SA, Goren AM et al. Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1. Biochemistry. 2015 Jan 20;54(2):208-220. https://doi.org/10.1021/bi5013047
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 "Bill" ; Walker, F. Ann. / Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1. In: Biochemistry. 2015 ; Vol. 54, No. 2. pp. 208-220.
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AU - Amoia, Angela M.

AU - Garrett, Sarah A.

AU - Goren, Allena M.

AU - Korte, Stephanie R.

AU - Zhang, Hongjun

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N2 - 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 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 "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.

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