Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: Stability and binding properties

Doris Wassenberg, Wolfgang Liebl, Rainer Jaenicke

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34 Scopus citations


Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75°C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition > 105°C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at ≃ 40°C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields ΔG(N→U) = 100 (± 5) kJ mol-1 for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol-1. At temperatures around the optimal growth temperature of T. maritima (t(opt) ≃ 80°C), the yield of refolding does not exceed 80%; the residual 20% are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with ΔH(b) = + 47 kJ mol-1 and ΔS(b) = + 257 J mol-1 K-1. Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) ≃ 0.3 μM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.

Original languageEnglish
Pages (from-to)279-288
Number of pages10
JournalJournal of Molecular Biology
Issue number2
StatePublished - 14 Jan 2000
Externally publishedYes


  • Folding
  • Ligand binding
  • Maltose-binding protein
  • Stability
  • Thermotoga maritima


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