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Spectroscopic and functional characterization of nitrophorin 7 from the blood-feeding insect Rhodnius prolixus reveals an important role of its isoform-specific N-terminus for proper protein function.
- Source :
-
Biochemistry [Biochemistry] 2007 Nov 20; Vol. 46 (46), pp. 13254-68. Date of Electronic Publication: 2007 Oct 24. - Publication Year :
- 2007
-
Abstract
- Nitrophorins (NPs) are a class of NO-transporting and histamine-sequestering heme b proteins that occur in the saliva of the bloodsucking insect Rhodnius prolixus. A detailed study of the newly described member, NP7, is presented herein. NO association constants for NP7 [KIII(eq)(NO)] reveal a drastic change when the pH is varied from 5.5 (reflecting the insect's saliva) to slightly above plasma pH (7.5) (>10(9) M-1 --> 4.0 x 10(6) M-1); thus, the protein promotes the storage of NO in the insect's saliva and its release inside the victim's tissues. In contrast to the other nitrophorins, NP1-4, histamine sequestering cannot be accomplished in vivo due to the low binding constant [KIII(eq)(histamine)] of 10(5) M-1 compared to the histamine concentration of 1-10 x 10(-9) M in the blood. A major part of this study deals with the N-terminus, 1Leu-Pro-Gly-Glu-Cys5 of NP7, which is not found in NP1-4. Since NP7 has not been isolated from the insects but was recognized in a cDNA library instead, the N-terminal site of signal peptidase cleavage upon protein secretion was predicted by the program SIGNALP [Andersen, J. F., Gudderra, N. P., Francischetti, I. M. B., Valenzuela, J. G., and Ribeiro, J. M. C. (2004) Biochemistry 43, 6987-6994]. In marked contrast to wild-type NP7, NP7(Delta1-3) exhibits a very high NO affinity at pH 7.5 [KIII(eq)(NO) approximately 10(9) M-1], suggesting that the release of NO in the plasma cannot efficiently be accomplished by the truncated form. Comparison of the reduction potentials of both constructs by spectroelectrochemistry revealed an average increase of +85 mV for various distal ligands bound to the heme iron when the 1Leu-Pro-Gly3 peptide was removed. However, 1H NMR and EPR spectroscopy show that the electronic properties of the FeIII cofactor are similar in both wild-type NP7 and NP7(Delta1-3). Further, thermal denaturation that revealed a higher stability of wild-type NP7 compared to NP7(Delta1-3), in combination with a homology model based on the NP2 crystal structure (rmsd = 0.39 A), suggests that interaction of the 1Leu-Pro-Gly3 peptide with the A-B and/or G-H loops is key for proper protein function.
- Subjects :
- Amino Acid Sequence
Animals
Electron Spin Resonance Spectroscopy
Hemeproteins physiology
Histamine chemistry
Histamine metabolism
Insect Proteins physiology
Kinetics
Ligands
Models, Molecular
Molecular Sequence Data
Nitric Oxide chemistry
Nitric Oxide metabolism
Nuclear Magnetic Resonance, Biomolecular
Protein Conformation
Protein Isoforms chemistry
Protein Isoforms physiology
Salivary Proteins and Peptides physiology
Sequence Alignment
Hemeproteins chemistry
Insect Proteins chemistry
Rhodnius metabolism
Salivary Proteins and Peptides chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 46
- Issue :
- 46
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17958381
- Full Text :
- https://doi.org/10.1021/bi7014986