Thermodynamics of tryptophan-mediated activation of the trp RNA-binding attenuation protein.
نویسندگان
چکیده
The trp RNA-binding attenuation protein (TRAP) functions in many bacilli to control the expression of the tryptophan biosynthesis genes. Transcription of the trp operon is controlled by TRAP through an attenuation mechanism, in which competition between two alternative secondary-structural elements in the 5' leader sequence of the nascent mRNA is influenced by tryptophan-dependent binding of 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 protein dynamics. 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, while analysis of the temperature dependence of the binding enthalpy by ITC revealed a negative heat capacity change larger than expected from surface burial, a hallmark of binding-coupled processes. Analysis of this excess heat capacity change using parameters derived from protein folding studies corresponds to the ordering of 17-24 residues per monomer of TRAP upon tryptophan binding. This result is in agreement with qualitative analysis of residue-specific broadening observed in TROSY NMR spectra of the 91 kDa oligomer. Implications for the mechanism of ligand-mediated TRAP activation through a shift in a preexisting conformational equilibrium and an induced-fit conformational change are discussed.
منابع مشابه
Thermodynamics of Tryptophan-Mediated Activation of the trp RNA-Binding Attenuation Protein (TRAP)
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Kinetic and thermodynamic analysis of the interaction between TRAP (trp RNA-binding attenuation protein) of Bacillus subtilis and trp leader RNA.
In Bacillus subtilis, expression of the tryptophan biosynthetic genes is regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein). TRAP has been shown to contain 11 identical subunits arranged in a symmetrical ring. Kinetic and thermodynamic parameters of the interaction between tryptophan-activated TRAP and trp leader RNA were studied. Res...
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عنوان ژورنال:
- Biochemistry
دوره 45 25 شماره
صفحات -
تاریخ انتشار 2006