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Determination of affinity constants for binding of Salmonella flagellar export proteins FliH and FliI
Author(s) -
Nguyen Viet Q.,
Kraft Lewis J.,
McMurry Jonathan L.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1006.3
Subject(s) - salmonella enterica , chemistry , flagellum , biophysics , microbiology and biotechnology , biochemistry , biology , gene , escherichia coli
Interaction of Salmonella enterica FliH with FliI is critically important to flagellar assembly, a Type III secretion process. FliH is a negative regulator of FliI, the export ATPase. The two have been shown qualitatively to stably associate. To quantitatively analyze the interaction, a saturation binding study was undertaken using an ELISA‐based approach after repurifying complexes of purified His‐tagged FliH equilibrated with varying amounts impure FLAG‐tagged FliI overproduced in a cell lysate. K D was determined to be 119 +/− 14 nM. No binding was detected with an N‐terminal FliH truncation (residues 1–102). Binding was also assayed using a C‐terminal FliH truncation (residues 99–235) shown to bind less stably than full‐length FliH in copurification assays. Results support the veracity of the assay. The present work shows that the FliH‐FliI association is comparable in affinity to that between FliH and the flagellar structural protein FliN and will serve as a basis for analyzing more complex protein assemblies such as FliN‐FliH‐FliI, illuminating the dynamics of flagellar export. This work was supported by grants from the NIH (R15GM080701), the Research Corporation (Cottrell College Science Award) and the KSU Mentor‐Protégé Program.