%0 Journal Article
%T Three hydrophobic amino acids in Escherichia coli HscB make the greatest contribution to the stability of the HscB-IscU complex
%A Anna K F¨¹z¨¦ry
%A Jenny J Oh
%A Dennis T Ta
%A Larry E Vickery
%A John L Markley
%J BMC Biochemistry
%D 2011
%I BioMed Central
%R 10.1186/1471-2091-12-3
%X 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.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 ¦¤¦¤Gb£¿ 5.7 kcal/mol, equivalent to a £¿ 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.Proteins containing iron-sulfur clusters play essential roles in electron-transfer, catalysis and other biochemical processes [1,2]. In eubacteria and in many eukaryotes, general iron-sulfur cluster biosynthesis is mediated by the multi-component ISC assembly system. Extensive biochemical and genetic studies [3-9] have shown that this process occurs through the assembly of a cluster on the scaffold protein IscU (Isu in yeast) followed by its transfer to a recipient apo-protein. The efficiency of the second step is greatly increased in the presence of HscA and HscB (Ssq1 and Jac1, respectively, in yeast), but the precise role of this chaperone system is not well understood [9-12].HscB is a 20 kDa J-type co-chaperone protein that regulates the ATP hydrolysis activity of HscA and targets IscU
%U http://www.biomedcentral.com/1471-2091/12/3