Assignment of the 1H NMR resonances of protein residues in close proximity to the heme of the nitrophorins: similarities and differences among the four proteins from the saliva of the adult blood-sucking insect Rhodnius prolixus.
The nuclear Overhauser effects (NOEs) observed between heme substituent protons and a small number of nearby protein side chain protons in the water-elimination Fourier transform NOE spectroscopy (WEFT-NOESY) spectra of high- and low-spin wild-type nitrophorin (NP) 2 and its ligand complexes have been analyzed and compared with those observed for the same complexes of wild-type NP3. These assignments were made on naturally abundant isotope samples, with the most useful protein side chains being those of Ile120, Leu122, and Leu132 for NP2 and NP3, and Thr121, Leu123, and Leu133 for NP1 and NP4. It is found that the NOEs observed are identical, with extremely similar protein side chain proton chemical shifts. This is strong evidence that the structure of NP3, for which no X-ray crystal structures are available, is essentially identical to that of NP2, at least near the heme binding pocket. Similarly, the NOEs observed between heme substituents and protein side chains for NP1 and NP4 also indicate that the structures of the protein having both A and B heme orientations are very similar to each other, as well as to the proteins with major B heme orientation of NP2 and NP3. These A and B connectivities can be seen, even though the two heme orientations have similar populations in NP1 and NP4, which complicates the analysis of the NOESY spectra. The histamine complex of wild-type NP2 shows significant shifts of the Leu132 side chain protons relative to all other ligand complexes of NP1-NP4 because of the perturbation of the structure near Leu132 caused by the histamine's side chain ammonium hydrogen bond to the Asp29 side chain carboxylate.
Department of Chemistry and Biochemistry, The University of Arizona, Tucson, AZ 85721-0041, USA. tatjana@email.arizona.edu
Tatiana K
TK
Shokhireva
F Ann
FA
Walker
D000818:Animals; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D012239:Rhodnius / Q000737:chemistry*; D012463:Saliva / Q000737:chemistry*; D012471:Salivary Proteins and Peptides / Q000737:chemistry*
D006420:Hemeproteins; D011522:Protons; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D006418:Heme