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Our work suggested that the binding site of IscU is in the C-terminal domain of HscB, and two different triple alanine substitutions ([L92A, M93A, F153A] and [E97A, E100A, E104A]) involving predicted binding site residues had detrimental effects on this interaction. However, the individual contribution of each substitution to the observed effect remains to be determined as well as the possible involvement of other residues in the proposed binding site.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>In the work reported here, we used isothermal titration calorimetry to characterize the affinity of single alanine HscB mutants for IscU, and subsequently confirmed our results with nuclear magnetic resonance spectroscopy. Alanine substitutions of L92, L96, and F153 severely impaired the ability of HscB to form a complex with IscU; substitutions of R87, R99, and E100 had more modest effects; and substitutions of T89, M93, E97, D103, E104, R152, K156, and S160 had only minor or no detectable effects.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>Our results show that the residues of HscB most important for strong interaction with IscU include three hydrophobic residues (L92, L96, and F153); in addition, we identified a number of other residues whose side chains contribute to a lesser extent to the interaction. Our results suggest that the triple alanine substitution at HscB positions 92, 96, and 153 will destabilize the HscB-IscU complex by \u0394\u0394<jats:italic>G<\/jats:italic><jats:sub>b<\/jats:sub>\u2245 5.7 kcal\/mol, equivalent to a \u2245 15000-fold reduction in the affinity of HscB for IscU. We propose that this triple mutant could provide a more definitive test of the functional importance of the HscB-IscU interaction in vivo than those used previously that yielded inconclusive results.<\/jats:p><\/jats:sec>","DOI":"10.1186\/1471-2091-12-3","type":"journal-article","created":{"date-parts":[[2011,1,26]],"date-time":"2011-01-26T19:20:46Z","timestamp":1296069646000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":36,"title":["Three hydrophobic amino acids in Escherichia coli HscB make the greatest contribution to the stability of the HscB-IscU complex"],"prefix":"10.1186","volume":"12","author":[{"given":"Anna K","family":"F\u00fcz\u00e9ry","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jenny J","family":"Oh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dennis T","family":"Ta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Larry E","family":"Vickery","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John L","family":"Markley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2011,1,26]]},"reference":[{"key":"312_CR1","doi-asserted-by":"publisher","first-page":"2","DOI":"10.1007\/s007750050002","volume":"5","author":"H Beinert","year":"2000","unstructured":"Beinert H: Iron-sulfur proteins: ancient structures, still full of surprises. 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