Leucine-induced dissociation of Escherichia coli Lrp hexadecamers to octamers.
Lrp is a global regulator of metabolism in Escherichia coli that helps cells respond to changes in environmental conditions. The action of Lrp as a transcriptional activator or repressor is sometimes affected when the medium contains exogenous leucine. In this study, we examined the thermodynamics of leucine binding to Lrp and to a leucine response mutant, Lrp-1, and leucine-induced dissociation of Lrp hexadecamer to leucine-bound octamer. The results of dynamic light-scattering and fluorescence measurements suggest that Lrp has two leucine-binding sites, one a high-affinity site and the other a low-affinity site that is coupled to the dissociation reaction. The Gibbs free energy change for leucine binding to the high-affinity site is about -7.0 kcal/mol. Binding of two leucine molecules to low-affinity sites on the hexadecamer or one leucine molecule to one octamer induces the dissociation of hexadecamer to leucine-bound octamer. The Gibbs free energy change for leucine binding to the low-affinity site was estimated to be in the range -4.66 to -5.03 kcal/mol for leucine binding to an octamer or -6.01 to -6.75 kcal/mol for leucine binding to a hexadecamer. The thermodynamic parameters derived from this study were used together with other data to estimate the distribution of free Lrp hexadecamer, octamer, leucine-bound hexadecamer, and leucine-bound octamer in cells. Mathematical modeling, employed to simulate modulation of Lrp action in response to growth conditions, gave results that are consistent with known patterns of Lrp action on different operons.
Chen
Shaolin
S
Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY 14853, USA.
Calvo
Joseph M
JM
D001667:Binding, Competitive; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005453:Fluorescence; D007930:Leucine / Q000494:pharmacology*; D051756:Leucine-Responsive Regulatory Protein; D008027:Light; D008962:Models, Theoretical; D009154:Mutation; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D014157:Transcription Factors; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C497278:Lrp protein, E coli; C497280:Lrp-1 protein, E coli; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein; D007930:Leucine