PMID: 9169618May 1, 1997Paper

Identification of critical residues in the colicin E9 DNase binding region of the Im9 protein

The Biochemical Journal
M J OsborneGeoffrey R Moore

Abstract

1H-15N NMR studies, in conjunction with mutagenesis experiments, have been used to delineate the DNase-binding surface of the colicin E9 inhibitor protein Im9 (where Im stands for immunity protein). Complexes were formed between the 15 kDa unlabelled E9 DNase domain and the 9.5 kDa Im9 protein uniformly labelled with 15N. Approx. 90% of the amide resonances of the bound Im9 were assigned and spectral parameters obtained from 1H-15N heteronuclear single quantum coherence (HSQC) spectra were compared with those for the free Im9 assigned previously. Many of the amide resonances were shifted on complex formation, some by more than 2 p.p.m. in the 15N dimension and more than 0.5 p.p.m. in the 1H dimension. Most of the strongly shifted amides are located on the surfaces of two of the four helices, helix II and helix III. Whereas helix II had already been identified through genetic and biochemical investigations as an important determinant of biological specificity, helix III had not previously been implicated in binding to the DNase. To test the robustness of the NMR-delineated DNase-binding site, a selection of Im9 alanine mutants were constructed and their dissociation rate constants from E9 DNase-immunity protein complexes quantif...Continue Reading

Citations

May 4, 2000·Protein Science : a Publication of the Protein Society·S B WhittakerG R Moore
Feb 7, 2002·Proceedings of the National Academy of Sciences of the United States of America·Stephen L SlatinDenis Duché
Oct 25, 2000·Protein Science : a Publication of the Protein Society·R BoetzelG R Moore

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