Thermodynamics of Tryptophan-Mediated Activation of the trp RNA-Binding Attenuation Protein

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• 作者：Craig A. McElroy ; Amanda Manfredo ; Paul Gollnick ; and Mark P. Foster
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：June 27, 2006
• 年：2006
• 卷：45
• 期：25
• 页码：7844 - 7853
• 全文大小：320K
• 年卷期：v.45,no.25(June 27, 2006)
• ISSN：1520-4995

文摘

The trp RNA-binding attenuation protein (TRAP) functions in many bacilli to control theexpression of the tryptophan biosynthesis genes. Transcription of the trp operon is controlled by TRAPthrough an attenuation mechanism, in which competition between two alternative secondary-structuralelements in the 5' leader sequence of the nascent mRNA is influenced by tryptophan-dependent bindingof TRAP to the RNA. Previously, NMR studies of the undecamer (11-mer) suggested that tryptophan-dependent control of RNA binding by TRAP is accomplished through ligand-induced changes in proteindynamics. We now present further insights into this ligand-coupled event from hydrogen/deuterium (H/D) exchange analysis, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC).Scanning calorimetry showed tryptophan dissociation to be independent of global protein unfolding, whileanalysis of the temperature dependence of the binding enthalpy by ITC revealed a negative heat capacitychange larger than expected from surface burial, a hallmark of binding-coupled processes. Analysis ofthis excess heat capacity change using parameters derived from protein folding studies corresponds to theordering of 17-24 residues per monomer of TRAP upon tryptophan binding. This result is in agreementwith qualitative analysis of residue-specific broadening observed in TROSY NMR spectra of the 91 kDaoligomer. Implications for the mechanism of ligand-mediated TRAP activation through a shift in apreexisting conformational equilibrium and an induced-fit conformational change are discussed.