Native N-terminus nitrophorin 2 from the kissing bug: similarities to and differences from NP2(D1A).

by Robert E. Berry*, Dhanasekaran Muthu, Tatiana K. Shokhireva, Sarah A. Garrett, Hongjun Zhang,and F. Ann Walker*Department of Chemistry and Biochemistry, The University of Arizona, P.O. Box 210041, Tucson, AZ85721-0041, USA (e-mail: berryr@email.arizona.edu, awalker@email.arizona.edu)Thefirstaminoacidofmaturenativenitrophorin2isasparticacid,andwhenexpressedinE.coli,thewild-type gene of the mature protein retains the methionine-0, which is produced by translation of thestart codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1Amutant, for which the Met0 is cleaved by the methionine aminopeptidase of E. coli (R. E. Berry, T. K.Shokhireva,I.Filippov,M. N.Shokhirev,H.Zhang,F. A.Walker,Biochemistry2007,46,6830).NativeN-terminus nitrophorin 2 ((DM0)NP2) has been prepared by employing periplasmic expression of NP2 inE. coli using the pelB leader sequence from Erwinia carotovora, which is present in the pET-26bexpression plasmid (Novagen). This paper details the similarities and differences between the threedifferent N-terminal forms of nitrophorin 2, (M0)NP2, NP2(D1A), and (DM0)NP2. It is found that theNMR spectra of high- and low-spin (DM0)NP2 are essentially identical to those of NP2(D1A), but therate and equilibrium constants for histamine and NO dissociation/association of the two are different.