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NMR studies of the dynamics of high-spin nitrophorins: comparative studies of NP4 and NP2 at close to physiological pH.

ABSTRACT: The ?-barrel nitrophorin (NP) heme proteins are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands. NO is bound to iron of the NPs and is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1-NP4, which have sequence similarities in two pairs, NP1 and NP4 (90% identical) and NP2 and NP3 (80% identical). The available crystal structures of NP4 have been used to propose that pH-dependent changes in the conformation of two loops between adjacent ?-strands at the front opening of the protein, the A-B and G-H loops, determine the rate of NO release. At pH 7.3, NP4 releases NO 17 times faster than NP2 does. In this work, the aqua complexes of NP4 and NP2 have been investigated by nuclear magnetic resonance (NMR) relaxation measurements to probe the pico- to nanosecond and micro- to millisecond time scale motions at two pH values, 6.5 and 7.3. It is found that NP4-OH2 is fairly rigid and only residues in the loop regions show dynamics at pH 6.5; at pH 7.3, much more dynamics of the loops and most of the ?-strands are observed while the ?-helices remain fairly rigid. In comparison, NP2-OH2 shows much less dynamics, albeit somewhat more than that of the previously reported NP2-NO complex [Muthu, D., Berry, R. E., Zhang, H., and Walker, F. A. (2013) Biochemistry 52, 7910-7925]. The reasons for this major difference between NP4 and NP2 are discussed.

SUBMITTER: Berry RE

PROVIDER: S-EPMC4303294 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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NMR studies of the dynamics of high-spin nitrophorins: comparative studies of NP4 and NP2 at close to physiological pH.

Berry Robert E RE Muthu Dhanasekaran D Yang Fei F Walker F Ann FA

Biochemistry 20141224 2

The β-barrel nitrophorin (NP) heme proteins are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands. NO is bound to iron of the NPs and is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1-NP4, which have sequence similarities in two pairs, NP1 and NP4 (90% identical) an ...[more]

PMID: 25486224

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Project description: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 (1)H 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 (1)H 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 (1)H{(13)C} heteronuclear multiple quantum correlation and (1)H-(1)H nuclear Overhauser effect spectroscopy spectra.

Project description:Cataract, a major cause of visual impairment worldwide, is the opacification of the eye's crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of ?-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the ?D-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of ?D-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human ?D-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human ?D-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human ?D-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human ?D-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.

Dataset Information

NMR studies of the dynamics of high-spin nitrophorins: comparative studies of NP4 and NP2 at close to physiological pH.

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NMR studies of the dynamics of high-spin nitrophorins: comparative studies of NP4 and NP2 at close to physiological pH.

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