Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus.
A nitric oxide transport protein (nitrophorin I) from the salivary glands of the blood-sucking bug Rhodnius prolixus has been expressed as an insoluble form in Escherichia coli, reconstituted with heme, and characterized with respect to NO binding kinetics and equilibria. NO binding and absorption spectra for recombinant nitrophorin I were indistinguishable from those of the insect-derived protein. The degree of NO binding, the rate of NO release, and the Soret absorption maxima for nitrophorin I were all pH dependent. The NO dissociation constant rose 9-fold over the pH range 5.0-8.3, from 0.19 x 10(-6) to 1.71 x 10(-6). The NO dissociation rate rose 2500-fold between pH 5.0 and pH 8.3, from 1.2 x 10(-3) to 3.0 s(-1). Thus, the NO association rate must also be pH dependent and reduced at pH 5.0 by approximately 280-fold. These factors are consistent with nitrophorin function: NO storage in the apparent low pH of insect salivary glands and NO release into the tissue of the insect's host, where vasodilation is induced. The reversible nature of NO binding, which does not occur with most other heme proteins, and the apparent kinetic control of NO release are discussed. We also report crystals of nitrophorin I that are suitable for structure determination by X-ray crystallography. The most promising crystal form contains two protein molecules in the asymmetric unit and diffracts beyond 2.0 A resolution.
Andersen
J F
JF
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Champagne
D E
DE
Weichsel
A
A
Ribeiro
J M
JM
Balfour
C A
CA
Dress
V
V
Montfort
W R
WR
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003460:Crystallization; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012239:Rhodnius; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D014961:X-Ray Diffraction
D002352:Carrier Proteins; D006420:Hemeproteins; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D009569:Nitric Oxide