Identification of the native N-terminus of the membrane attaching ferriheme protein nitrophorin 7 from Rhodnius prolixus

Knipp, Markus; Soares, Rodrigo Pedro; Pereira, Marcos H.

doi:10.1016/j.ab.2012.02.010

Cited by 9 publications

(3 citation statements)

References 20 publications

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“…Upon maturation, a major difference of NP7 compared to all other NPs is its extended N-terminus ( Figure S3 ), which is characterized by the extension of the tripeptide Leu1–Pro2–Gly3 8 , 56 . It should be mentioned that the N-terminus of the recombinant NP7 contains an additional Met0 residue originating from the start codon of the expression system.…”

Section: Resultsmentioning

confidence: 99%

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

et al. 2015

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Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

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Section: Resultsmentioning

confidence: 99%

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

et al. 2015

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“…9 The majority of the Lys residues cluster at the protein's surface opposite to the opening to the heme pocket and can thus attach to negatively charged phospholipid membranes. 8,10,11 Due to its bipolar charge distribution, NP7 tends to form oligomers and insoluble aggregates in solution. 12−17 Therefore, solution-state NMR studies of NP7 were hampered by broad resonances due to slow molecular tumbling rates of transient aggregates and thus accelerated T 2 relaxation.…”

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“…In contrast to NP1–4, NP7 contains a high number of positively charged Lys residues, which results in the high pI of 9.2 compared to 6.1–6.5 for NP1–4 . The majority of the Lys residues cluster at the protein’s surface opposite to the opening to the heme pocket and can thus attach to negatively charged phospholipid membranes. ,, Due to its bipolar charge distribution, NP7 tends to form oligomers and insoluble aggregates in solution. − Therefore, solution-state NMR studies of NP7 were hampered by broad resonances due to slow molecular tumbling rates of transient aggregates and thus accelerated T 2 relaxation . Recently, crystallization conditions for NP7 were established that yield small crystals; however, certain ill-defined electron density in the heme pocket complicates the refinement process.…”

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Expression, Purification, and Solid-State NMR Characterization of the Membrane Binding Heme Protein Nitrophorin 7 in Two Electronic Spin States

et al. 2013

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The nitrophorins (NPs) comprise a group of NO transporting ferriheme b proteins found in the saliva of the blood sucking insect Rhodnius prolixus . In contrast to other nitrophorins (NP1-4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of (13)C,(15)N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with S = 1/2 and S = 0 at the ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center.

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Preparation of nitrophorin 7(Δ1–3) from Rhodnius prolixus without start–methionine using recombinant expression in Escherichia coli

Risse

Taing

Knipp

2014

Analytical Biochemistry

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Identification of the native N-terminus of the membrane attaching ferriheme protein nitrophorin 7 from Rhodnius prolixus

Cited by 9 publications

References 20 publications

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

Expression, Purification, and Solid-State NMR Characterization of the Membrane Binding Heme Protein Nitrophorin 7 in Two Electronic Spin States

Preparation of nitrophorin 7(Δ1–3) from Rhodnius prolixus without start–methionine using recombinant expression in Escherichia coli

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