NDLI: NMR investigations of nitrophorin 2 belt side chain effects on heme orientation and seating of native N-terminus NP2 and NP2(D1A)

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NMR investigations of nitrophorin 2 belt side chain effects on heme orientation and seating of native N-terminus NP2 and NP2(D1A)

Content Provider	Springer Nature Link
Author	Berry, Robert E. Muthu, Dhanasekaran Shokhireva, Tatiana K. Garrett, Sarah A. Goren, Allena M. Zhang, Hongjun Walker, F. Ann
Copyright Year	2013
Abstract	Nitrophorin 2 (NP2), one of the four NO-storing and NO-releasing proteins found in the saliva of the blood-sucking bug Rhodnius prolixus, has a more ruffled heme and a high preference for a particular heme orientation (B) compared with nitrophorin 1 and nitrophorin 4, which show not a preference (A to B ratio of approximately 1:1), suggesting that it fits more tightly in the β-barrel protein. In this work we have prepared a series of “belt” mutants of NP2(D1A) and (ΔM0)NP2 aimed at reducing the size of aromatic or other residues that surround the heme, and investigated them as the high-spin aqua and low-spin N-methylimidazole complexes. The belt mutants included Y38A, Y38F, F42A, F66A, Y85A, Y85F, Y104A, I120T, and a triple mutant of NP2(D1A), the F42L, L106F, I120T mutant. Although I120 has been mainly considered to be a distal pocket residue, CδH3 of I120 lies directly above the heme 3-methyl, at 2.67 Å, of heme orientation B, or the 2-vinyl of A, and it thus plays a role as a belt mutant, a role that turns out to be extremely important in creating the strong favoring of the B heme orientation [A to B ratio of 1:14 for NP2(D1A) or 1:12 for (ΔM0)NP2]. The results show that the 1D 1H NMR spectra of the high-spin forms are quite sensitive to changes in the shape of the heme binding cavity. The single mutation I120T eliminates the favorability of the B heme orientation by producing a heme A to B orientation ratio of 1:1, whereas the single mutation F42A reverses the heme orientation from an A to B ratio of 1:14 seen for NP2(D1A) to 10:1 for NP2(D1A,F42A). The most extreme ratio was found for the triple mutant of NP2(D1A), NP2(D1A,F42L,L105F,I120T), in which the A to B ratio is approximately 25:1, a ΔG change of about −3.5 kcal/mol or −14.1 kJ/mol with respect to NP2(D1A). The seating of the heme is modified as well in that mutant and in several others, by rotations of the heme by up to 4° from the seating observed in NP2(D1A), in order to relieve steric interactions between a vinyl β-carbon and a protein side chain, or to fill a cavity created by replacing a large protein side chain by a much smaller one; the latter was observed for all tyrosine to alanine mutants. These relatively small changes in seating have a measurable effect on the NMR spectra of the mutants, but are indeed minor in terms of overall seating and reactivity of the NP2(D1A) protein. The 1H NMR resonances of the hemin substituents of the low-spin N-methylimidazole complexes of NP2(D1A,F42L,L105F,I120T) as well as NP2(D1A,I120T), NP2(D1A,Y104A), and NP2(D1A,F42A) have been assigned using natural abundance 1H{13C} heteronuclear multiple quantum correlation and 1H–1H nuclear Overhauser effect spectroscopy spectra.
Starting Page	577
Ending Page	593
Page Count	17
File Format	PDF
ISSN	09498257
Journal	JBIC Journal of Biological Inorganic Chemistry
Volume Number	19
Issue Number	4-5
e-ISSN	14321327
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2013-11-30
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Heme Nuclear magnetic resonance Protein engineering Site-directed Mutagenesis Structure–function relationship Thermodynamics Biochemistry Microbiology
Content Type	Text
Resource Type	Article
Subject	Biochemistry Inorganic Chemistry

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NMR Investigations of Nitrophorin 2 Belt Side Chain Effects on Heme Orientation and Seating of Native N-terminus NP2 and NP2(D1A)

NMR Investigations of Nitrophorin 2 Belt Side Chain Effects on Heme Orientation and Seating of Native N-terminus NP2 and NP2(D1A)

NMR Studies of Nitrophorin Distal Pocket Side Chain Effects on the Heme Orientation and Seating of NP2 as Compared to NP1

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NMR investigations of nitrophorin 2 belt side chain effects on heme orientation and seating of native N-terminus NP2 and NP2(D1A)

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