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

Journal Article · · Biochemistry
DOI:https://doi.org/10.1021/bi5013047· OSTI ID:1169372
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  1. Univ. of Arizona, Tucson, AZ (United States). Dept. of Chemistry and Biochemistry
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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. Lastly, 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.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Organization:
National Institutes of Health (NIH); USDOE
Grant/Contract Number:
W-31-109-Eng-38; HL054826; HL062969; T32 GM008659; RR07707.
OSTI ID:
1169372
Journal Information:
Biochemistry, Vol. 54, Issue 2; ISSN 0006-2960
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 1 work
Citation information provided by
Web of Science

References (52)

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