title: NMR studies of the dynamics of nitrophorin 2 bound to nitric oxide.
abstract: The Rhodnius nitrophorins are β-barrel proteins of the lipocalin fold with a heme protruding from the open end of the barrel. They are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands, where NO is bound to iron. NO 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. At pH 7.3, NP4 releases NO 17 times faster than NP2 does, as measured by stopped-flow kinetics. A number of crystal structures of the least abundant protein, NP4, are available. These structures have been used to propose that two loops between adjacent β-strands at the front opening of the protein, the A-B and G-H loops, determine the rate of NO release. To learn how the protein loops contribute to the release of NO for each of the nitrophorins, the dynamics of these proteins are being studied in our laboratory. In this work, the NP2-NO complex has been investigated by nuclear magnetic resonance relaxation measurements to probe the picosecond-to-nanosecond and microsecond-to-millisecond time scale motions at three pH values, 5.0, 6.5, and 7.3. It is found that at pH 5.0 and 6.5, the NP2-NO complex is rigid and only a few residues in the loop regions show dynamics, while at pH 7.3, somewhat more dynamics, particularly of the A-B loop, are observed. Comparison to other lipocalins shows that all are relatively rigid, and that the dynamics of lipocalins in general are much more subtle than those of mainly α-helical proteins.
authors.affiliation: Department of Chemistry and Biochemistry, The University of Arizona , 1306 East University Boulevard, Tucson, Arizona 85721-0041, United States.
authors.forename: Dhanasekaran
authors.initials: D
authors.lastname: Muthu
authors.forename: Robert E
authors.initials: RE
authors.lastname: Berry
authors.forename: Hongjun
authors.initials: H
authors.lastname: Zhang
authors.forename: F Ann
authors.initials: FA
authors.lastname: Walker
mesh_terms: D000818:Animals; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D006420:Hemeproteins; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D009569:Nitric Oxide